Introduction

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Hypoxia-induced pulmonary hypertension (PH) is associated with persistent vasoconstriction and structural remodeling of pulmonary vessels responsible for increased medial thickness of muscular arteries, peripheral extension of arterial muscularization, and increased matrix deposition (1). Subsequently, loss of distal functional pulmonary arteries results in increased pulmonary vascular resistance.

The angiogenic factor vascular endothelial growth factor (VEGF), a homodimeric 34- to 42-kD heparin-binding glycoprotein, is a peptide mitogen specific for endothelial cells that fulfills its function by binding to flt-1 and KDR/flk-1, two highly specific tyrosine kinase receptors expressed almost exclusively on endothelial cells (2). Accumulating evidence indicates that stimulation of angiogenesis under hypoxic and ischemic conditions involves upregulation of VEGF and its receptors by hypoxia (3, 4). In the lung, VEGF is primarily and abundantly expressed by epithelial cells (5). Previous studies performed in chronically hypoxic rats have found increases in lung VEGF and VEGF receptors expression (6, 7). Increased VEGF protein and transcripts have also been found in the plexiform lesions of vessels from patients with primary PH (7). However, results obtained in our laboratory as well as in others failed to demonstrate increased lung VEGF expression during development of hypoxic PH (8, 9). Moreover, in the experimental model of monocrotaline-induced PH, which is associated with intense vascular remodeling, a dramatic decrease in lung VEGF messenger RNA expression was observed (8, 10). Specifically, the role of endogenous VEGF in the development of PH and attendant vascular remodeling remains unclarified. In addition to its well-known angiogenic properties, VEGF has been shown to protect against endothelial vascular injury and to improve endothelial function (11). Our previous finding of impaired endothelium-dependent relaxation in chronic hypoxic PH (14) invited an investigation of whether lung VEGF overexpression can protect against hypoxic PH and alter the development of pulmonary vascular remodeling.

Gene therapy may be a valuable therapeutic approach in PH. Several studies using intratracheal administration of adenovirus vectors have shown that transgene expression is located mainly in epithelial cells (15, 16). Using this route of administration, previous studies demonstrated that adenoviral-mediated gene transfer of human endothelial nitric oxide synthase (eNOS) in rats was associated with a reduction in acute pulmonary vasoconstriction (17). We therefore reasoned that overexpression of a secreted and diffusible form of VEGF (VEGF₁₆₅) in epithelial cells after adenoviral-mediated gene transfer may affect endothelial cell behavior and protect against pulmonary vascular remodeling during development of hypoxic PH. To investigate this hypothesis, we used a previously described adenovirus vector containing an expression cassette with the cytomegalovirus (CMV) early/intermediate promoter/enhancer driving the human VEGF₁₆₅ complementary DNA (cDNA) (Ad.VEGF) (18).

In the first part of this study, we evaluated the efficiency of gene transfer after a single intratracheal instillation of Ad.VEGF by measuring levels of VEGF protein in bronchoalveolar fluid and serum after various doses of the adenovirus vector and at various times after the instillation. We also evaluated the effect of Ad.VEGF on pulmonary vessel permeability by measuring the extravascular accumulation of radiolabeled albumin corrected for lung blood weight. In the second part of the study, we assessed pulmonary hemodynamics, right ventricular hypertrophy, and pulmonary vascular remodeling in rats pretreated with intratracheal administration of Ad.VEGF 2 d before the start of a 2-wk exposure to normoxia or hypoxia. Finally, to investigate the mechanisms of the protective effect of Ad.VEGF on the development of hypoxic PH, we measured eNOS activity in lung tissue and examined pulmonary vasoreactivity using isolated lungs from normoxic and chronically hypoxic rats pretreated with Ad.VEGF.

	Materials and Methods

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Recombinant Adenovirus Vectors

The replication-deficient vector Ad.VEGF is an E1a, partial E1b, partial E3 Ad vector with an expression cassette in the E1 position containing the CMV immediate early promoter/enhancer driving the cDNA for the 165-residue form of human VEGF (18). Ad.Null (similar to Ad.VEGF, but with no gene in the expression cassette) was used as a control vector. All adenovirus vectors were propagated in 293 cells, purified by CsCl gradient centrifugation, dialyzed, and stored at 80°C as previously described (19). The titer of each viral stock was determined by plaque assay in 293 cells.

Animals and Delivery of Adenovirus Vectors to the Lungs

Wistar rats (200 to 250 g body weight) were used for all studies. All animal care and procedures were in accordance with institutional guidelines. Ad.VEGF, or Ad.Null as the control, was diluted before use with sterile saline, pH 7.4, in a final volume of 150 µl. Rats were anesthetized with intraperitoneal ketamine (7 mg/ 100 g) and xylazine (1 mg/100 g). Intratracheal instillation of 150 µl/rat of diluted Ad.VEGF or Ad.Null was performed using a standard procedure, as previously described (16).

Evaluation of Gene Transfer

Human VEGF protein detection in serum and bronchoalveolar lavage fluid. To evaluate gene transfer efficiency, serum and bronchoalveolar lavage fluid (BALF) levels of human VEGF protein were measured in normoxic rats 5 d after administration with various doses of Ad.VEGF (10⁸ to 5 × 10⁹ plaque-forming units [pfu]).

Human VEGF protein was also measured in serum and BALF from normoxic rats 2, 5, 10, and 17 d after administration with Ad.VEGF or Ad.Null (10⁸ pfu).

After intraperitoneal administration of pentobarbital (60 mg/ kg), blood samples were drawn from the abdominal aorta. Sera obtained after centrifugation of clotted blood samples at 2,000 rpm for 10 min were stored at 20°C. Bronchoalveolar lavage (BAL) was performed immediately after blood sampling. A total of 25 ml of warm phosphate-buffered saline (PBS) was used for each rat. Five aliquots of 5 ml were separately instilled, recovered, and pooled. The supernatants of BALF samples spun at 2,000 rpm, 4°C, for 15 min, were stored at 20°C.

Enzyme-linked immuno-assay (ELISA) detection of human VEGF was carried out on serum and BALF samples using Quantikine (R&D Systems, France). The assay was carried out according to the supplier's instructions, and absorbance at 490 nm was determined on a plate reader.

Histologic evaluation of inflammation after gene transfer. To evaluate the inflammatory response after adenovirus administration, histologic examination was also performed in normoxic rats at various times after treatment with Ad.VEGF or Ad.Null (10⁸ pfu) and 5 d after various doses of Ad.VEGF (as explained earlier).

Immediately after BAL, the lungs were removed and fixed by infusion of neutral buffered formaldehyde into the trachea at 25 cm H₂O. After routine processing and paraffin-embedding, multiple sections from each lobe were stained with hematoxylin and eosin. The inflammatory response was analyzed using a previously described empiric semiquantitative scale (16) based on inflammatory cell type and location (alveoli, bronchi, blood vessels) and on the presence of edema and hemorrhage. Epithelial damage in bronchi, bronchioles, or alveoli was scored 0 to 4 (absent to severe, respectively). Extension of inflammation was also scored 0 to 4 as follows: 0, none; 1, small patchy areas involved; 2, < 10% of section area; 3, 10 to 50%; 4, > 50%.

Effect of Ad.VEGF on pulmonary vascular permeability. To evaluate the effect of Ad.VEGF on vascular permeability, rats were administered Ad.VEGF or Ad.Null (10⁸ pfu) and then exposed to normoxia (n = 12) or hypoxia (n = 12). The extravascular accumulation of radiolabeled albumin was measured 5 d after gene transfer. The technique has been previously described (20). In brief, blood was obtained from donor rats and centrifugated. The pellet was incubated at 37°C with [⁵¹Cr] for 30 min and then washed twice with saline. Red blood cells were resuspended in plasma. A mixture of a radiolabeled [¹²⁵I]albumin (1 µCi in 200 µl) and blood containing the [⁵¹Cr]-labeled red blood cells (5 to 10 µCi in 300 µl) was injected into the jugular vein of rats anesthetized with ketamine and xylazine. At 1 h later, the chest was opened and a blood sample obtained by cardiac puncture before removal of the lung. The lung and blood samples were weighed and counted in a multichannel scintillation counter. Extravascular accumulation was calculated as whole-lung [¹²⁵I] counts minus intravascular-lung [¹²⁵I] counts, where intravascular [¹²⁵I] counts = (blood [¹²⁵I] activity) × ([⁵¹Cr] counts in lung/blood [⁵¹Cr]). The extravascular-lung [¹²⁵I] count was then divided by intravascular radiolabeled protein concentration (blood [¹²⁵I] activity) and lung blood weight ([⁵¹Cr] count in lung/blood [⁵¹Cr] activity) to calculate a protein leak index that should minimize the effects of differences in vascular surface area and relative lung mass.

Effect of Ad.VEGF Pretreatment on Hypoxic PH

To examine the effect of Ad.VEGF administration on hypoxic PH, Ad.VEGF or Ad.Null was administered intratracheally 2 d before the beginning of exposure to normoxia or hypoxia. Hemodynamic measurements and assessment of right ventricular hypertrophy and pulmonary vascular remodeling were performed after 15 d of continuous exposure to normoxia or hypoxia (17 d after gene transfer).

Exposure of rats to chronic hypoxia. Rats were exposed to chronic hypoxia (10% O₂) in a ventilated chamber (500-liter volume; Flufrance, Cachan, France), as previously described (14).

Hemodynamic measurements and assessment of right ventricular hypertrophy. At the end of the 2-wk exposure to normoxia or hypoxia, the rats were anesthetized using an intramuscular injection of ketamine (7 mg/100 g) and xylazine (1 mg/100 g). After exposure of the right jugular vein, a polyvinyl catheter was inserted and manipulated through the right ventricle (RV) into the pulmonary artery. A polyethylene catheter was inserted into the right carotid artery. Pulmonary and systemic arterial pressures were measured under normoxic breathing conditions, immediately after insertion of the catheters, using Gould P 23 ID transducers coupled to pressure modules and a Gould TA 550 multichannel recorder. Only pulmonary artery pressures successfully recorded within 30 min of catheter insertion were taken into account. In some animals, blood was also sampled from the systemic artery catheter for hematocrit measurement. Finally, after an intraperitoneal injection of pentobarbital sodium (60 mg/kg), the thorax was opened, the heart was excised and weighed, and the ratio of the weight of the right ventricular free wall to the weight of the septum plus the left ventricular free wall was estimated.

Assessment of pulmonary vascular remodeling. After BAL was performed as previously described, the lungs were fixed in the distended state by infusion of formalin into the trachea. A midsaggital slice of the right lung was processed for paraffin embedding and sections stained with hematoxylin-phloxin-saffron and orcein-picroindigo-carmine. In each rat, a total of 50 to 65 intraacinar vessels accompanying either alveolar ducts or alveoli were analyzed by an observer blinded to the treatment. Each vessel was categorized as muscular, partially muscular, or nonmuscular as previously described (21). The external diameter (the distance between and including the two external elastic laminae intersected by the diameter) and medial thickness (the distance from the luminal surface of the internal elastic lamina to the abluminal surface of the external lamina) were recorded for all muscular and partially muscular arteries. Normalized wall thickness (WT_N) was calculated using the following formula: WT_N (%) = ([2 × medial thickness] ^ [external diameter]) × 100. The ratio of the number of distal arteries (50 to 200 µm) over the number of alveoli was also assessed on each section.

Pharmacologic Studies in Isolated Lungs from Normoxic and Chronically Hypoxic Rats

Isolated lungs were perfused through a pulmonary arterial cannula using a peristaltic pump at a constant flow of 0.05 ml/g body weight/min with a recirculated physiologic salt solution containing Ficoll (4 g/100 ml, type 70; Sigma Chemical Co., St. Louis). At the start of some experiments, meclofenamate (3.2 µM) was added to the perfusate to achieve complete cyclooxygenase inhibition. Each lung preparation was used for only one of the following procedures.

Effect of exogenous VEGF in isolated lungs from normoxic and chronically hypoxic rats (2 wk). We examined the effect of exogenous VEGF or its vehicle in preconstricted isolated lungs from normoxic (n = 4) and chronically hypoxic (n = 4) rats. After a 30-min equilibration period, the lungs were preconstricted with the endoperoxide analog U-46619, diluted in a 20-ml volume of physiologic salt solution, and infused into the pulmonary arterial line at a constant rate of 50 pmol/min. Pulmonary artery pressure increased gradually in response to U-46619 and did not reach a plateau. Increasing doses (10, 50, 100, and 250 ng) of recombinant human VEGF (Sigma) diluted in distilled water were injected as 50-µl boluses at 3-min intervals into the pulmonary arterial line during U-46619 infusion when the increase in pulmonary arterial pressure reached 6 to 7 mm Hg. These experiments were performed without meclofenamate.

Assessment of vascular reactivity in lungs from rats pretreated with Ad.VEGF or Ad.Null (10⁸ pfu) before exposure to normoxia or chronic hypoxia. The effect of the endothelium-dependent vasodilator agent ionophore A23187 and the vasoconstrictor response to endothelin (ET)-1 were assessed in isolated lungs from rats pretreated with Ad.VEGF or Ad.Null (10⁸ pfu) 2 d before the beginning of a 2-wk exposure to normoxia or hypoxia. These experiments were performed in the presence of meclofenamate as previously described (14).

The vasodilator effect of ionophore A23187 or its vehicle was tested in lungs preconstricted with U-46619 as described earlier. Ionophore diluted in solutions of increasing concentrations was injected as 50-µl bolus in the perfusate reservoir at 3-min intervals to obtain increasing final concentrations in the recirculating perfusate (10^8.5 to 10⁷ mol/liter).

The vasoconstrictor effect of ET-1 was tested in lungs under baseline conditions. After 30 min of equilibration, ET-1 was injected into the arterial line as a 50-µl bolus containing 300 pmol. The pressor response to ET-1 was measured 20 min after administration. To assess the ability of endothelial cells to produce nitric oxide (NO) in response to ET-1, we also examined the effects of the nitric oxide synthesis (NOS) inhibitor nitro-L-arginine methylester (L-NAME) (5 × 10⁴ mol/liter) on ET-1-induced vasoconstriction. The antagonist was added to the perfusate reservoir 10 min before administration of ET-1.

Assessment of eNOS Activity in Lungs from Rats Pretreated with Ad.VEGF or Ad.Null (10⁸ pfu)

eNOS activity was assessed in lung tissue from rats that had received intratracheal Ad.VEGF or Ad.Null (10⁸ pfu) 2 d before the beginning of a 2-wk exposure to either normoxia or hypoxia. Rats without adenovirus administration (sham) were also studied. On removal, lungs were quickly frozen in liquid nitrogen. Tissue was homogenized on ice using an Ultraturax homogenizer in 4 vol of buffer containing 50 mmol/liter Tris-HCl (pH 7.4), 0.1 mmol/liter ethylenediaminetetraacetic acid (EDTA), 0.1 mmol/liter ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), 0.1% 2-mercaptoethanol, 1 µmol/liter leupeptin, 1 µmol/ liter pepstatin A, and 1 mmol/liter phenylmethylsulfonyl fluoride as previously described (22). The homogenate was centrifuged at 100,000 × g for 1 h at 5°C. To remove soluble proteins, the pellet was resuspended in homogenization buffer containing 1 mol/liter KCl and allowed to stand on ice for 5 min before centrifugation at 100,000 × g for 30 min at 5°C. The supernatant fraction was discarded and the pellet (membrane fraction) resuspended in homogenization buffer containing the detergent CHAPS (20 mmol/ liter), 1 mol/liter KCl, and glycerol (10% vol/vol) and allowed to stand on ice for 30 min before centrifugation at 100,000 × g for 30 min at 5°C.

Activity of eNOS in the resultant supernatant fraction (membrane fraction) was determined by measuring the Ca²⁺-dependent conversion of L-[³H]arginine to L-[³H]citrulline in the reaction mixture. The enzyme extract (25 µl) was added to 200 µl of the reaction mixture containing 50 mmol/liter Tris-HCl (pH 7.4), 10 µmol/liter tetrahydrobiopterin, 1 mmol/liter dithiothreitol, 10 µg/ ml calmodulin, 4 µmol/liter flavin adenine dinucleotide, 4 µmol/ liter Flavin Mononucleotide, 2 µmol/liter L-arginine, 1 Kcpm/µl L-[³H]arginine, and 1 mmol/liter nicotinamide adenine dinucleotide phosphate with or without 1 mmol/liter CaCl₂. After 40 min of incubation at 37°C, the reaction was stopped with 2 ml of a solution containing 20 mmol/liter Na acetate pH 5.5, 1 mmol/liter L-citrulline, 2 mmol/liter EDTA, and 0.2 mmol/liter EGTA. The mixture was applied to a 1-ml Dowex AG 50WX8 column and L-[³H]citrulline was eluted with 2 ml of distilled water. The radioactivity in the eluate was measured by liquid scintillation spectroscopy. The concentration of protein in the enzyme extract was determined according to Lowry.

Statistical Analysis

All results are expressed as means ± standard error of the mean (SEM). A two-way analysis of variance (ANOVA) was performed to compare the effect of Ad.VEGF versus Ad.Null pretreatment in normoxic and hypoxic rats, followed by Fisher's test or nonparametric Mann-Whitney test to compare Ad.VEGF and Ad.Null pretreatment for each condition of oxygenation when interaction was significant. To compare the degrees of pulmonary vessel muscularization in the two groups of rats, vessels were ordinally classified as nonmuscular, partially muscular, and muscular. Comparison of muscularization between two groups was performed separately at the alveolar duct and wall levels using a nonparametric Mann-Whitney test. To compare the effect of ionophore A23187 versus its vehicle in isolated rat lungs, two-way ANOVA with repeated measurements was performed; followed, because interaction was significant, by nonparametric Mann- Whitney test to compare drug versus vehicle at each dose.

	Results

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Evaluation of In Vivo Gene Transfer in Normoxic Rats

Dose-dependent protein expression and inflammatory response 5 d after adenovirus administration. As shown in Table 1, as little as 10⁸ pfu of Ad.VEGF produced detectable amounts of human VEGF protein in BALF. With increasing amounts of Ad.VEGF, human VEGF protein increased in a dose-dependent manner in BALF and became detectable in serum. The intensity of the inflammatory response also varied in a dose-dependent manner. With 10⁸ pfu of either Ad.VEGF or Ad.Null, inflammation was characterized by patchy infiltrates of mononuclear cells, most of which were macrophages. No cell damage, edema, or hemorrhage was observed. Higher doses of Ad.VEGF produced a more diffuse inflammatory response, accompanied in some cases by mild cell damage in the alveolar and bronchiolar epithelium, edema, and hemorrhage. These changes likely reflected the tissue response to the viral vectors and were consistent with previous findings (15, 16). On the basis of this dose-response experiment, 10⁸ pfu was the dose selected for further experiments because it caused minimal inflammatory responses but significant human VEGF protein production in lungs without detectable levels in serum. After treatment with Ad.Null (10⁸ pfu), no human VEGF was detected in BALF or serum.

Time-course of protein expression after adenovirus administration (10⁸ pfu). Expression of human VEGF protein in BALF samples was detectable on Day 2 and peaked on Day 5 after treatment with Ad.VEGF (10⁸ pfu) (Figure 1). On Day 17, human VEGF protein was still detectable in BALF from five of eight rats.

View larger version (10K): [in this window] [in a new window]   Figure 1.   Time-course of human VEGF protein expression in BALF from normoxic rats treated with Ad.VEGF (108 pfu). At 0, 2, 5, 10, and 17 d after intratracheal administration of Ad.VEGF (108 pfu), BAL was performed using warm PBS with a final volume of 25 ml for each rat. The human VEGF protein level in BALF was determined by ELISA. n indicates number of animals studied at each time.

Effect of Ad.VEGF (10⁸ pfu) treatment on pulmonary vascular permeability. There was a trend for an increase in pulmonary vascular permeability in hypoxic as compared with normoxic rats, but differences did not reach statistical significance. Transvascular protein escape measured at maximum of protein expression was not altered by Ad.VEGF pretreatment. Indeed, in rats exposed to normoxia, protein leak index was not significantly increased 5 d after gene transfer in Ad.VEGF- as compared with Ad.Null-treated rats (1.62 ± 0.33 versus 1.20 ± 0.15, respectively; not significant [NS]). When rats were exposed to hypoxia immediately after gene transfer, protein leak index also did not differ between Ad.VEGF- and Ad Null-pretreated rats (2.09 ± 0.18 and 1.64 ± 0.43, respectively; NS).

Effects of Ad.VEGF Pretreatment on Chronic Hypoxic PH

Administration of either Ad.VEGF or Ad.Null (10⁸ pfu) was well tolerated in animals studied during the 17 d. No deaths or symptoms of respiratory failure were observed in rats exposed to normoxia or chronic hypoxia.

Hemodynamic measurements and assessment of right ventricular hypertrophy. In rats exposed to normoxia for 17 d after adenovirus administration, pulmonary artery pressure (Table 2) and RV weight over either body weight or left ventricle (LV) plus septum (LV+S) weight (Figure 2) did not differ between Ad.VEGF- and Ad.Null-treated animals and were similar to values previously reported in normoxic rats.

View larger version (25K): [in this window] [in a new window]   Figure 2.   Ratio of RV to LV plus septum weight (RV/LV+S) in rats exposed to normoxia (left) or chronic hypoxia (2 wk; right) and pretreated with either Ad.VEGF or Ad.Null. Pretreatment with Ad-VEGF significantly attenuated RV/LV+S in hypoxic rats but had no effect on normoxic rats. *P < 0.05 for comparison with corresponding values obtained in rats pretreated with Ad.Null.

Exposure to hypoxia for 15 d after adenovirus pretreatment was associated with an increase in pulmonary artery pressure and development of right ventricular hypertrophy in Ad.VEGF and Ad.Null rats. However, in chronically hypoxic rats, pulmonary artery pressure was significantly lower after Ad.VEGF than after Ad.Null pretreatment (P < 0.05), whereas systemic arterial pressure and heart rate were similar (Table 2). Right ventricular hypertrophy as assessed by RV weight over either body weight or LV+S weight (Figure 2) was also less marked in chronically hypoxic rats pretreated with Ad.VEGF than in those given Ad.Null (P < 0.001), whereas LV weight was similar in both groups. After exposure to hypoxia, hematocrit was also similar in Ad.VEGF- and Ad.Null-pretreated rats (46 ± 0.1% and 45 ± 0.1%, respectively). Final body weight did not differ between these two groups.

Measurement of human VEGF in BALF. Human VEGF protein was still detectable in the BALF from 7 out of 13 of these chronically hypoxic rats studied 17 d after administration of Ad.VEGF. Values ranged from 3 to 1,942 pg/ml.

Structural remodeling of distal pulmonary vessels. In each rat, a total of 35 to 65 intraacinar vessels were examined to determine the percentage of nonmuscularized and partially and totally muscularized vessels according to the accompanying airway (alveolar duct or alveolar wall). In rats exposed to normoxia for 17 d after adenovirus administration, muscularization of distal vessels did not differ between Ad.VEGF- and Ad.Null-treated animals and were similar to values previously reported in normoxic rats (Figure 3A). Pretreatment with Ad.VEGF partially prevented muscularization of distal pulmonary arteries in response to hypoxia at both the alveolar duct and the alveolar wall level (Figure 3B; P < 0.001). Moreover, as compared with control animals (Ad.Null) similarly exposed to hypoxia, the normalized wall thickness of muscularized and partially muscularized arteries was markedly reduced in Ad.VEGF-pretreated rats (Figure 4; P < 0.001; and Figure 5).

View larger version (22K): [in this window] [in a new window]   View larger version (25K): [in this window] [in a new window]   Figure 3.   Percentage of non- (NM), partially (PM), or fully muscularized (M) vessels according to the accompanying airway. A total of 50 to 65 intraacinar vessels was analyzed in each lung from rats exposed to normoxia (A) or 2-wk hypoxia (B) and pretreated with either Ad.VEGF or Ad.Null. After exposure to chronic hypoxia, muscularization at alveolar duct or alveolar wall level was significantly lower after Ad.VEGF than Ad.Null pretreatment (P < 0.001, nonparametric Mann-Whitney test on ordinally classified vessels). Pretreatment with Ad-VEGF did not affect muscularization of pulmonary vessels in normoxic rats.

View larger version (24K): [in this window] [in a new window]   Figure 4.   Normalized wall thickness measured in muscular or partially muscular arteries (external diameter of 50 to 200 µm) in lungs from rats exposed to chronic hypoxia (2 wk) and pretreated with either Ad.VEGF (n = 11) or Ad. Null (n = 8). A total of 50 to 65 intraacinar vessels was analyzed in each rat.

View larger version (107K): [in this window] [in a new window]   Figure 5.   Pulmonary vascular remodeling illustrated by representative photomicrographs of pulmonary vessels from rats exposed to chronic hypoxia (2 wk) and pretreated with either Ad.VEGF (a and b) or Ad.Null (c and d). Sections of 5 µm thickness were cut for light microscopy and stained with hematoxylin-phloxin-saffron. Original magnification: ×40: 15 µm represents 50 mm.

The number of distal vessels (50 to 200 µm) was counted on 10 to 20 sections per rat and normalized for the number of alveoli. After 15 d of hypoxia, no significant difference was observed between the Ad.VEGF-treated group (12.2 ± 0.9 arteries per 100 alveoli) and the Ad.Null group (11.0 ± 0.3), suggesting the absence of new vessel development.

Pharmacologic Studies in Isolated Lungs

Response to exogenous VEGF in isolated lungs from normoxic and chronically hypoxic rats. As compared with vehicle alone, in isolated lungs from either normoxic or chronically hypoxic rats, increasing doses of recombinant human VEGF had no effect on the increase in perfusion pressure induced by continuous infusion of U-46619 (data not shown).

Assessment of pulmonary vascular reactivity in lungs from rats pretreated with Ad.VEGF or Ad.Null (10⁸ pfu). Mean baseline pulmonary artery pressure was similar in lungs from rats exposed to 17 d of normoxia after Ad.VEGF or Ad.Null administration.

Exposure to 2 wk of hypoxia (10% O₂) after Ad.VEGF or Ad.Null administration was associated with an increase in mean baseline perfusion pressure as compared with lungs from normoxic rats. However, in lungs from hypoxic rats, perfusion pressure was significantly lower after Ad.VEGF than after Ad.Null pretreatment (Table 3; P < 0.05). Moreover, in lungs from normoxic or hypoxic rats, the pressor response after 1 or 10 min of U-46619 infusion (50 pmol/min) was less marked after Ad.VEGF than after Ad.Null pretreatment (P < 0.001). In subsequent experiments performed to assess the vasodilator effect of ionophore A23187, the rate of U-46619 infusion in lungs from Ad.Null-pretreated rats was decreased to 25 pmol/min to obtain approximately the same pressure increase in both groups of lungs before ionophore administration.

In lungs from normoxic rats, ionophore A23187 elicited a dose-dependent decrease in perfusion pressure that was similar after Ad.VEGF or Ad.Null pretreatment (Figure 6A). In contrast, ionophore A23187 did not modify the perfusion pressure increase in response to U-46619 in lungs from hypoxic rats pretreated with Ad.Null, whereas it stabilized perfusion pressure despite maintenance of U-46619 infusion (Figure 6B) in lungs from Ad.VEGF-pretreated rats similarly exposed to hypoxia.

View larger version (15K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   Figure 6.   Vasodilatory response to ionophore A23187 during a continuous infusion of U-46619 as indicated by pressure on y axis versus concentration of ionophore on x axis, in lungs from rats exposed to normoxia (A) or chronic hypoxia (B) and pretreated with either Ad.Null or Ad.VEGF. Ionophore A23187 ( filled circles) or vehicle (diluted ethanol; open circles) was administered into the perfusate reservoir as 50 µl of increasing doses (final concentration: 108.5 to 107 mol/liter) separated by time intervals of 3 min. * P < 0.05, ** P < 0.01 for comparison with corresponding time control values measured with vehicle. n = 5 in each experiment.

Under conditions of baseline tone, ET-1 (300 pmol) induced an increase in perfusion pressure (Figure 7). This increase was similar in lungs from normoxic rats pretreated with Ad.Null or Ad.VEGF. After exposure to hypoxia, the increase in perfusion pressure elicited by ET-1 was larger than in lungs from normoxic rats and also more marked in lungs from Ad.Null than in lungs from Ad.VEGF rats (P < 0.05). Pretreatment with the NO synthesis inhibitor L-NAME (5 × 10⁴ mol/liter) potentiated the pressor response to ET-1 in lungs from both groups of normoxic rats as well as in lungs from hypoxic rats pretreated with Ad.VEGF, but had no effect in lungs from rats pretreated with Ad.Null and similarly exposed to hypoxia. After L-NAME pretreatment, the pressor response to ET-1 did not differ between lungs of the two groups of hypoxic rats.

View larger version (22K): [in this window] [in a new window]   Figure 7.   Increase in baseline perfusion pressure in response to ET-1 (300 pmol) in isolated lungs from rats exposed to normoxia (left) and to chronic hypoxia (right) and pretreated with either Ad.Null or Ad.VEGF. Vasopressor effect of ET was measured in the presence of L-NAME (5 × 104 mol/ liter), an inhibitor of NOS, or its vehicle (H2O), which were added to the perfusate reservoir 20 min before the injection of ET-1 (n = 5 in each experiment).

Effect of Ad.VEGF Treatment on eNOS Activity

Results of the Ca²⁺-dependent NOS activity assessed in the membrane fraction of lung tissue after 2 wk of exposure to normoxia or hypoxia are shown in Figure 8. There was no significant effect of hypoxia as compared with normoxia on eNOS activity, which also did not differ between Ad.Null-pretreated rats and those without adenovirus administration (sham) (two-way ANOVA). However, eNOS activity was increased in Ad.VEGF- as compared with Ad.Null-pretreated (P < 0.05) or sham (P < 0.01) rats.

View larger version (19K): [in this window] [in a new window]   Figure 8.   Values of eNOS activity in lung tissue from rats exposed to normoxia or chronic hypoxia (2 wk) without (Sham; open bars) or with pretreatment with either Ad.VEGF (stippled bars) or Ad.Null ( filled bars). Numbers in columns indicate numbers of animals studied. Values from Ad.Null and Ad.VEGF differed significantly (P < 0.01, Fisher's test) but no differences between Ad.Null and sham or between normoxia and hypoxia, as well as no interaction between pretreatments and exposure, were observed (two-way ANOVA).

	Discussion

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Our findings show that adenoviral-mediated lung VEGF overexpression, which had no effect on the pulmonary circulation during normoxia, protected rats exposed to chronic hypoxia against development of PH. Values were lower for pulmonary arterial pressure, right ventricular hypertrophy, and distal vessel muscularization in hypoxic rats pretreated with Ad.VEGF as compared with rats pretreated with the control vector Ad.Null. Our results also suggest that this protective effect of VEGF on the pulmonary circulation during exposure to chronic hypoxia may be at least partially related to an improvement in endothelial function. Indeed, Ad.VEGF pretreatment was associated with increased lung eNOS activity, partial restoration of the vasodilator response to the endothelium-dependent agent ionophore A23187, and marked blunting of ET-1- induced vasoconstriction.

VEGF is now considered a potent angiogenic mediator that can be overexpressed in target tissues via either a replication-deficient adenovirus vector or naked DNA (23- 25). In various models of myocardial or peripheral ischemia, as well as in humans with peripheral arteriopathy, VEGF has been shown to promote revascularization of ischemic tissues by stimulating collateral vessel development (26, 27). Although the mechanisms leading to collateral vessel growth in response to VEGF are not well understood (28), it is now widely accepted that hypoxia is a major stimulus for angiogenic factor expression and activity. In addition to its mitogenic effects on endothelial cells, VEGF has also been shown to improve endothelial function and to diminish systemic artery remodeling (11, 13, 29). However, the effects of VEGF on pulmonary vessels have not been examined, particularly during exposure to hypoxia, which leads to pulmonary vascular remodeling and subsequent development of PH. Increased lung VEGF expression may be desirable during hypoxia-induced PH, not only to promote the development of new pulmonary vessels but also to improve endothelial function. Moreover, the pulmonary arteries affected by remodeling during PH are in close proximity to epithelial cells from distal airways. Transfer of the human VEGF₁₆₅ gene to these cells by intratracheal administration of an adenoviral vector (17, 19) may therefore provide a means of exposing pulmonary vessels to high levels of a diffusible and secreted form of VEGF.

In the present study, adenoviral transfer allowed efficient local overexpression of human VEGF₁₆₅ in rat lungs. After intratracheal administration of Ad.VEGF (10⁸ pfu), the VEGF₁₆₅ protein was detected in BALF for about 17 d, with a concentration peak on Day 5. No VEGF was detectable in the serum with this low dose of adenovirus, suggesting that the risk of VEGF diffusion or expression in other organs was minimal. As shown by our histologic study, administration of 10⁸ pfu of Ad.VEGF or Ad.Null caused only mild inflammation in lungs from both normoxic and hypoxic rats. Only small patchy areas of macrophage infiltrates were observed, with no epithelial cell damage, edema, or hemorrhage. Moreover, measurement of transvascular protein escape at time of peak expression did not provide evidence for an increased vascular permeability in lungs from rats pretreated with this low dose of Ad.VEGF and subsequently exposed to normoxia or hypoxia.

The main finding from our study is that VEGF overexpression in lung tissue did not affect the pulmonary circulation in normoxic conditions but attenuated the development of PH and right ventricular hypertrophy in rats exposed to chronic hypoxia. Pulmonary artery pressure and RV weight were lower in rats pretreated with Ad.VEGF than in hypoxic control rats pretreated with Ad.Null, whereas systemic arterial pressure and LV weight were similar. Attenuation of PH was not related to an effect of VEGF overexpression on hypoxia-induced polycythemia, because hematocrit did not differ between the two groups of adenovirus-infected animals. The development of hypoxic PH is associated with smooth-muscle cell hypertrophy and hyperplasia in normally muscularized arteries and with the appearance of new smooth-muscle cells in nonmuscularized segments of the intraacinar circulation. In addition to decreases in PH and right ventricular hypertrophy, reductions in the percentage of muscularized arteries at the alveolar duct and wall level and in muscularized artery wall thickness were seen in hypoxic rats pretreated with Ad.VEGF. In the aim to explain this protective effect of VEGF overexpression on development of PH, we questioned whether these effects could be due to a direct vasodilator effect of VEGF on the pulmonary circulation. However, in contrast to effects shown in coronary arteries (13), we did not observe any direct vasodilator effect of VEGF in the rat pulmonary circulation. Bolus administration of the recombinant protein did not reverse the vasoconstrictor effect of U-46619 in isolated lungs from either normoxic or chronically hypoxic rats. We also questioned whether lung VEGF overexpression could be associated with an increase in the number of pulmonary arteries. Indeed, newly formed collateral arteries have previously been demonstrated in response to VEGF after occlusion of large systemic arteries in the limb circulation or in the heart (2, 24, 25, 27). In case of development of newly formed pulmonary arteries, one could speculate that VEGF overexpression, even transient, provides long-term protection against PH. In the present study, lung VEGF overexpression was not associated with a significant increase in the number of distal vessels, as assessed by counting the number of distal vessels over the number of alveoli. These results, therefore, differ from those obtained in experimental animal models of local ischemia after treatment with VEGF. It is now established that arteriogenesis depends on a pre-existing network of arterioles that is present in most tissues, especially in the vascular periphery of the limb circulation and the heart (28). Because such an arteriolar network is not present in the lung, arteriogenesis may not develop in response to angiogenic factors in the pulmonary vascular tree. Together, these observations demonstrate that the beneficial effect of VEGF overexpression on PH development was due to diminished vascular remodeling, with no evidence for an increase in the number of pulmonary arteries.

It is now well known that focal vascular injury and impaired endothelial function are important features of PH that lead to enhanced platelet endothelial cell interactions and contribute to thrombosis and smooth-muscle proliferation. Recently, numerous studies have suggested that in addition to its mitogenic effect on endothelial cells, VEGF may act as an endothelial cell "survivor factor" (13, 30). In systemic arteries, increasing VEGF bioavailability at sites of endothelial injury has been shown to accelerate endothelial repair and to limit neointima formation (29, 30). Moreover, VEGF overexpression within the vascular wall has been shown to restore endothelial-dependent relaxation and to protect against vasoconstriction (11).

In the present study, VEGF overexpression improved endothelium-dependent vasodilation and blunted vasoreactivity to the constrictor agents U-46619 and ET-1 in lungs from chronically hypoxic rats. We have previously shown that endothelial function is impaired in the pulmonary circulation from rats exposed to chronic hypoxia (14). The vasodilator response to acetylcholine or ionophore A23187, two endothelium-dependent agents, is abolished in isolated lungs from rats previously exposed for 3 wk to severe hypoxia. In the present study, we found that VEGF overexpression did not affect endothelium-dependent vasodilation to ionophore A23187 in lungs from normoxic rats but partially restored it after exposure to chronic hypoxia. Moreover, the vasoconstrictor response to ET-1, which was not affected by VEGF overexpression in lungs from normoxic rats, was significantly increased in lungs from hypoxic rats. However, in rats similarly exposed to hypoxia, the pressor response to ET-1 was less marked in Ad.VEGF- than in Ad.Null-pretreated rats. It is noteworthy that pressor response to ET-1 in Ad.VEGF-pretreated hypoxic rats was potentiated after L-NAME pretreatment, reaching the same magnitude as in lungs from Ad.Null rats exposed to a similar degree and duration of hypoxia. In conjunction with the later results, we also found that lung eNOS activity was enhanced in lungs from animals treated with Ad.VEGF as compared with those treated with Ad.Null. Altogether, these findings demonstrate that VEGF overexpression in the lungs led to protection of endothelial function and enhanced release of endothelial NO formation which may have contributed to attenuation of hypoxia-induced pulmonary vascular remodeling.

The reasons for the impaired NO-mediated vasodilation during chronic hypoxia-induced PH are not yet completely understood inasmuch as eNOS protein expression and activity have been shown to be unchanged or increased in lung tissue from chronically hypoxic rats (22, 31). These paradoxical findings clearly appeared in the present study showing abolition of ionophore A23187- induced vasodilation despite no decrease in eNOS activity. Thus, in chronic hypoxic PH, eNOS, although present in increased amounts, may produce insufficient NO to oppose hypoxia-induced constriction and associated vascular remodeling. Our previous results showing protection against hypoxia-induced PH by continuous inhalation of NO are consistent with this hypothesis (21). Despite our observation that acute supplementation of L-arginine improves endothelium-dependent NO formation in lungs from chronically hypoxic rats, long-term improvement of endothelial NO formation could not be achieved by chronic L-arginine treatment (32). Numerous studies have shown that VEGF increases production of NO in systemic arteries. However, the mechanism of this effect remains presently unclear. VEGF may interfere with eNOS at several levels, including interaction with other membrane proteins such as caveolin, protein trafficking, or substrate availability in the vicinity of the enzyme (33, 34). In a recent study, VEGF has also been shown to increase eNOS expression via activation of the KDR receptor tyrosine-kinase and a downstream protein kinase C signaling pathway (35). The signaling pathway between VEGF and eNOS as well as other regulators of eNOS is probably not specific for the pulmonary circulation.

Our finding that VEGF overexpression in the lungs obtained by means of adenovirus-mediated gene transfer improves endothelial function and attenuates development of PH and vascular remodeling in a rat model of hypoxic PH may have clinical implications regarding the treatment of PH. However, further studies are needed to determine whether VEGF overexpression can also reverse established PH and whether it also plays a beneficial role in other models of PH. The principal limitation of adenoviral vector-mediated transfer may be the limited duration of VEGF expression in lung tissue. However, rather than a limitation, this feature may be an advantage in some situations where expression during only a few weeks may provide both adequate therapeutic efficacy and limited side effects. Development of second-generation adenoviral vectors will provide new tools to prolong the duration of transgene expression.