Ambrisentan reduces pulmonary arterial hypertension but does not stimulate alveolar and vascular development in neonatal rats with hyperoxic lung injury

Abstract

Ambrisentan, an endothelin receptor type A antagonist, may be a novel therapeutic agent in neonatal chronic lung disease (CLD) by blocking the adverse effects of the vasoconstrictor endothelin-1, especially pulmonary arterial hypertension (PAH)-induced right ventricular hypertrophy (RVH). We determined the cardiopulmonary effects of ambrisentan treatment (1–20 mg·kg⁻¹·day⁻¹) in neonatal rats with CLD in 2 models: early treatment during continuous exposure to hyperoxia for 10 days and late treatment starting on day 6 in rat pups exposed postnatally to hyperoxia for 9 days, followed by a 9-day recovery period in room air. Parameters investigated included survival, lung and heart histopathology, right ventricular function, fibrin deposition, and differential mRNA expression in the lungs. In the early treatment model, we investigated the role of nitric oxide synthase (NOS) inhibition with N^ω-nitro-l-arginine methyl ester (l-NAME; 25 mg·kg⁻¹·day⁻¹) during ambrisentan treatment. In the early treatment model, ambrisentan improved survival with reduced lung fibrin and collagen III deposition, arterial medial wall thickness, and RVH. These changes were not affected by l-NAME administration. Ambrisentan did not reduce the influx of macrophages and neutrophils or prevent reduced irregular elastin expression. In the late treatment model, ambrisentan diminished PAH, RVH, and right ventricular peak pressure, demonstrating that RVH is reversible in the neonatal period. Alveolarization and vascularization were not affected by ambrisentan. In conclusion, ambrisentan prolongs survival and reduces lung injury, PAH, and RVH via a NOS-independent mechanism but does not affect inflammation and alveolar and vascular development in neonatal rats with CLD.

lung immaturity and subsequent treatment for respiratory distress after premature birth with resuscitation, mechanical ventilation, and supplemental oxygen makes the premature lung highly susceptible to injury. Proinflammatory mediators produced by the injured lung may interfere with signaling pathways required for normal lung development and may lead to neonatal chronic lung disease (CLD) or bronchopulmonary dysplasia (BPD) (5). The hallmark of CLD is alveolar enlargement due to an arrest in alveolar and vascular development. Serious complicating factors in the perinatal period are inflammation and oxidative stress, and at later stages pulmonary arterial hypertension (PAH) that ultimately leads to right ventricular hypertrophy (RVH) and heart failure (1, 5, 21–23). PAH is characterized by persistent vasoconstriction and structural remodeling of the pulmonary blood vessels with increased proliferation of vascular smooth muscle cells, leading to a decrease in blood vessel lumen. This process ultimately leads to high mortality due to right heart failure in children and adults (1, 2, 40, 42).

Endothelin-1 (ET-1) is a potent vasoconstrictor and proinflammatory agent and induces PAH and cardiac hypertrophy (14, 18, 25). ET-1 expression is highest in normal rat lung (14) and is detected in endothelial, airway epithelial and alveolar type 2 cells. In rats with bleomycin-induced pulmonary fibrosis, ET-1 is expressed in inflammatory cells (33). In endothelial cells, ET-1 is synthesized as a 203-amino acid prepropeptide, which is cleaved to a 38-amino acid peptide (big ET-1) by furin convertase (9). Big ET-1 is transformed into ET1_1–21 by endothelin-converting enzyme-1 (ECE-1), chymase, or non-ECE metalloproteases. ET1_1–21 is secreted and exerts its biological effects after binding to endothelin receptor type A (ET_A), localized on smooth muscle and epithelial cells, fibroblasts, macrophages, and cardiac myocytes, or endothelin receptor type B (ET_B), localized on fibroblasts, macrophages, and endothelial, epithelial, and smooth muscle cells (13, 24, 37, 45). Binding of ET-1 to ET_A or ET_B receptors on vascular smooth muscle cells results in vasoconstriction and proliferation and migration of smooth muscle cells (31, 34, 37). Binding of ET-1 to ET_B receptors on endothelial cells is associated with beneficial effects on inflammatory lung disease by inducing prostacyclin and nitric oxide (NO) release, which leads to vasodilation and reduced apoptosis and ECE-1 expression in endothelial cells and mediates pulmonary clearance of ET-1 from the circulation through endothelial cell reuptake (8, 12, 17). ET-1 expression increases under many pathological conditions, including hypoxia, hyperoxia, ischemia, shear stress, and bleomycin-induced pulmonary fibrosis, and in the presence of cytokines, growth factors, and thrombin. NO, prostacyclin, and atrial natriuretic peptides reduce ET-1 expression (13, 14, 16, 33). The adverse effects of ET-1 can be blocked by endothelin-1 receptor antagonists. We studied the specific ET_A antagonist ambrisentan as a novel therapeutic strategy in severe BPD (7, 30, 43). This experimental approach will preserve the potential beneficial effects of endothelin binding to ET_B receptors on endothelial cells and block the adverse effects of endothelin binding to pulmonary ET_A receptors, especially inflammation and PAH-induced RVH. In ventilated premature lambs with respiratory distress syndrome, short-term treatment with the ET_A receptor antagonist BQ-123 attenuates pulmonary vascular resistance (19). In neonatal mice, treatment with the ET_A receptor antagonist BQ-610 attenuates hypoxia-induced lung injury (3) but does not improve alveolar enlargement (32). The effect of ET receptor blockage on alveolar enlargement in mammalian neonatal and adult lung are conflicting, showing reduced alveolar enlargement in cigarette smoke extract-induced lung injury in adults (7) but no improvement of hypoxia-induced neonatal alveolar enlargement (32).

Because the potential of long-term treatment of neonatal hyperoxia-induced lung injury (experimental CLD) with ET receptor antagonists is unknown, we investigated the cardiopulmonary effects of ambrisentan treatment in newborn rats with experimental CLD induced by exposure to 100% oxygen. We used two different treatment strategies: a prophylactic strategy (early concurrent treatment) and a more clinically relevant strategy, in which treatment was started after lung injury was induced in a lung injury recovery model (late treatment and recovery). Because ambrisentan only blocks ET_A receptors and preserves beneficial effects of ET_B receptor signaling on endothelial cells via endothelial nitric oxide synthase (eNOS), we studied the role of the NO-cGMP pathway by specific inhibition of NOS activity with N^ω-nitro-l-arginine methyl ester (l-NAME). Early concurrent treatment with ambrisentan exerted beneficial effects on survival, coagulation, alveolar septal thickness, collagen III deposition, and PAH-induced RVH. These beneficial effects were not affected by l-NAME administration. In the injury recovery model, ambrisentan reduced PAH-induced RVH. Some results of these studies have been reported previously as an abstract (47).

MATERIALS AND METHODS

Full details of the methods are available from the author.

Animals

The research protocol was approved by the Institutional Animal Care and Use Committee of the Leiden University Medical Center. In the early concurrent treatment model, neonatal rat pups were pooled and distributed over four experimental groups (n = 12), i.e., an oxygen, oxygen-ambrisentan, oxygen-l-NAME, and oxygen-l-NAME-ambrisentan group, and four room air-exposed control groups injected with saline, ambrisentan, and/or l-NAME. In the late treatment and recovery model, newborn rat pups were distributed among two experimental groups (n = 12), i.e., an oxygen and oxygen-ambrisentan group, and two room air-exposed control groups injected with either saline or ambrisentan. In a separate experiment, lung tissue was collected from neonates on days 1, 3, 6, and 10 and from adult rats (6 mo) for RT-PCR (n = 8). Oxygen concentration, body weight, evidence of disease, and mortality were monitored daily.

Early Concurrent Treatment

Pups were continuously exposed to 100% oxygen for 10 days. From day 2 onward, pups received subcutaneous injections of ambrisentan (a gift from Gilead Sciences, Foster City, CA) in 100 μl of 0.9% saline, 100 μl of 0.9% saline (age-matched control), 25 mg·kg⁻¹·day⁻¹ of l-NAME (Sigma, St Louis, MO) in 0.9% saline, or l-NAME plus ambrisentan in 0.9% saline (100 μl, subcutaneously). A concentration of 25 mg·kg⁻¹·day⁻¹ of l-NAME in 0.9% saline completely abolishes the beneficial effects of apelin, which are dependent on eNOS activation, in experimental BPD (11). Lung and heart tissue were collected on day 10. To find the optimal dose for treatment of experimental BPD with ambrisentan, we performed a pilot experiment in which hyperoxia-exposed rat pups were treated with 1–20 mg·kg⁻¹·day⁻¹ of ambrisentan or saline (n = 6). Because hyperoxia-induced lung injury results in severe CLD with persistent PAH-induced RVH (10, 11) and ambrisentan is a very potent treatment option for PAH and RVH in adults (2), we used the ratio of right ventricle to left ventricle (RV/LV) in histological sections of the heart as a readout. We found that pups exposed to hyperoxia developed RVH, which could be attenuated (5 mg/kg twice a day or 10 mg/kg once a day) or completely reversed (10 mg/kg twice a day) by treatment with ambrisentan (Fig. 1). Therefore, experiments were performed with 20 mg·kg⁻¹·day⁻¹ of ambrisentan (10 mg/kg twice a day). Separate experiments were performed for 1) histology (n = 8), 2) frozen lung (n = 10) and heart tissue (n = 12), and 3) collection of bronchoalveolar lavage fluid (n = 12). Because l-NAME had no effect on the beneficial effects of early ambrisentan treatment, we did not include l-NAME treatment in the late treatment and recovery experiments.

Fig. 1.

Pilot experiment to find the optimal dose of ambrisentan for treatment of experimental bronchopulmonary dysplasia (BPD) by determining right ventricular hypertrophy (RVH) depicted as right ventricular (RV)/left ventricular (LV) wall thickness ratio on day 10 after early concurrent treatment (n = 6) in room air (RA)-exposed pups injected daily with saline (open bar) and oxygen (O₂)-exposed pups injected daily with saline (solid bar) or ambrisentan [1, 5, and 10 mg·kg⁻¹·day⁻¹ once a day and 5 and 10 mg·kg⁻¹·day⁻¹ twice a day (2×); shaded bars]. Data are means ± SE. *P < 0.05; ***P < 0.001 vs. age-matched O₂-exposed controls. ΔP < 0.05; ΔΔP < 0.01 vs. RA control.

Late Treatment and Recovery

Lung injury and recovery were investigated by exposing pups to hyperoxia for 9 days, followed by recovery in room air for 9 days. After 6 days of hyperoxia, daily injections with 0.9% saline or 20 mg·kg⁻¹·day⁻¹ of ambrisentan in 0.9% saline were started and continued throughout the 9-day recovery period in room air. Lung and heart tissue were collected on day 9, after 9 days of hyperoxic lung injury (n = 8), and on day 18, after 9 days of recovery in room air (n = 8).

Tissue Preparation

Lungs and heart were snap-frozen in liquid nitrogen for real-time RT-PCR or fibrin deposition assays or fixed in formalin for histology studies (10, 11).

Histology

Formalin-fixed, paraffin-embedded, 4-μm-thick heart and lung sections were stained with hematoxylin and eosin (HE). Furthermore, lung tissue sections were Hart's stained to visualize elastin (38) by incubation for 24 h in a solution containing 20 ml of Resorcin-fuchsin solution (no. X877.2; Carl Roth, Karlsruhe, Germany), 200 ml of 70% ethanol, and 4 ml of hydrochloric acid. After four washes in double-distilled water, sections were counterstained with tartrazine solution containing 0.5 g of tartrazine (no. 86310; Sigma-Aldrich), 200 ml of double-distilled water, and 0.5 ml of acetic acid. Lung sections were also immunostained with anti-ED-1 (monocytes and macrophages; 1:5), anti-MPO (myeloperoxidase; no. RB-373-A1; Thermo Fisher Scientific, Fremont, CA; diluted 1:1,500), anti-ASMA (α-smooth muscle actin; no. A2547; Sigma-Aldrich; diluted 1:10,000), anti-vWF (von Willebrand factor; no. A0082; Dako Cytomation, Glostrup, Denmark; diluted 1:4,000), or anti-COL3A1 (collagen III; no. ab7778; Abcam, Cambridge, UK; diluted 1:3,000). Hereafter, sections were stained with EnVision-horseradish peroxidase (Dako), using the chromogenic substrate NovaRed as recommended by the manufacturer (Vector, Burlingame, CA), and counterstained briefly with hematoxylin using standard methods (10, 11). For morphometry of the lung, an eye piece reticle with a coherent system of 21 lines and 42 points (Weibel type II ocular micrometer; Olympus, Zoeterwoude, The Netherlands) was used (46). We used different (immuno)histochemically stained lung sections for each quantification, except for alveolar crest and pulmonary arterial medial wall thickness, which were determined on the same ASMA-stained section. To investigate alveolar enlargement in experimental CLD, we measured the number of alveolar crests to exclude potential effects of heterogenous alveolar development. The number of alveolar crests (49), determined on lung sections stained immunohistochemically for ASMA, were assessed in 10 nonoverlapping fields at ×400 magnification for each animal and were normalized at the field level. The density of ED-1-positive monocytes and macrophages or MPO-positive neutrophilic granulocytes was determined in the alveolar compartment by counting the number of cells per field. Results are expressed as cells per square millimeter. In each animal 20 fields in one section were studied at ×400 magnification. Pulmonary alveolar septal thickness was assessed in HE-stained lung sections at ×400 magnification by averaging 100 measurements per 10 representative fields. Capillary density was assessed in lung sections stained for vWF at ×200 magnification by counting the number of vessels per field. At least 10 representative fields per experimental animal were investigated. Results are expressed as relative number of vessels per square millimeter. Pulmonary arterial medial wall thickness was assessed in lung sections stained for ASMA at ×1,000 magnification by averaging at least 10 vessels with a diameter of <30 μm per animal. Arterial medial wall thickness was calculated from the formula %wall thickness = (2 × wall thickness/external diameter) × 100. Extracellular collagen III deposition was quantified on lung sections stained immunohistochemically for COL3A1 with the NIH ImageJ program by calculating the relative area positive for collagen III at ×300 magnification in 10 nonoverlapping fields. Elastin was quantified on lung sections stained histochemically for elastin with the NIH ImageJ program by calculating the relative area positive for elastin at ×200 magnification in 10 nonoverlapping fields. Fields containing large blood vessels or bronchioli were excluded from the analysis. Thickness of the RV and LV free walls and interventricular septum (IVS) was assessed in a transversal HE-stained section taken halfway on the long axis at ×40 magnification by averaging 6 measurements per structure. Quantitative morphometry was performed on a microscope (model BX-41TF, equipped with digital camera UC30; Olympus) by two independent researchers, blinded to the treatment strategy, using the NIH ImageJ program (10, 49).

Fibrin Detection Assay

Quantitative fibrin deposition was determined in lung tissue homogenates by Western blotting (10, 46). Lung tissue homogenates for quantitative fibrin deposition by Western blotting were pretreated as described previously (46). Tissue samples, dissolved in reducing sample buffer (10 mM Tris, pH 7.5, 2% SDS, 5% glycerol, 5% β-mercaptoethanol, and 0.4 mg/ml bromophenol blue) were subjected to SDS-PAGE (7.5% gel; 5% stacking gel) and blotted onto polyvinylidene difluoride membrane (Immobilon-FL; Millipore, Bedford, MA). The 56-kDa fibrin β-chains were detected with monoclonal 59D8 antibody (Oklahoma Medical Research Foundation, Oklahoma City, OK; diluted 1:1,000), infrared-labeled with goat-anti-mouse secondary antibody (IRDye 800CW; Licor Biosciences, Lincoln, NE; diluted 1:5,000), and quantified using an infrared detection system (Odyssey infrared imaging system; Licor Biosciences). Fibrin deposition was quantified by using rat fibrin as a reference.

Bronchoalveolar Lavages and Protein Assay

Total protein content in lung lavages was determined as an indicator of vascular leakage using a standard protein assay (DC protein assay; Bio-Rad, Veenendaal, The Netherlands) according to the manufacturer's instructions, using bovine serum albumin (fraction V; Roche Diagnostics, Almere, The Netherlands), as previously described (10).

Real-Time RT-PCR

Total RNA isolation from lung and heart tissue homogenates (RNA-Bee; Tel-Test, Bio-Connect, Huissen, The Netherlands), first-strand cDNA synthesis (SuperScript Choice System; Life Technologies, Breda, The Netherlands), and real-time quantitative PCR, using β-actin as a housekeeping gene reference, were performed on an ABI Prism 7900 HT Fast Real-Time PCR system (Applied Biosystems, Carlsbad, CA) of the Leiden Genome Technology Center as described previously (46). Primers are listed in Table 1.

Table 1.

Sequences of oligonucleotides for forward and reverse primers for real-time RT-PCR

Gene Product	Forward Primer	Reverse Primer
Amphiregulin	5′-TTTCGCTGGCGCTCTCA-3′	5′-TTCCAACCCAGCTGCATAATG-3′
ECE-1	5′-GCCCACCCTGGGTCTCA-3′	5′-AGCACCAGACCTGTGCGAAT-3′
ET-1	5′-TGTGCTCACCAAAAAGACAAGAA-3′	5′-GGTACTTTGGGCTCGGAGTTC-3′
ETA	5′-CACGACCAAGTTCATGGAGTTTT-3′	5′-AGGGCATGCAGAAGTAGAATCC-3′
ETB	5′-CAGGATTCTGAAGCTCACCCTTT-3′	5′-TCCAAAACCAGCAAAAAACTCA-3′
IL-6	5′-ATATGTTCTCAGGGAGATCTTGGAA-3′	5′-TGCATCATCGCTGTTCATACAA-3′
PAI-1	5′-AGCTGGGCATGACTGACATCT-3′	5′-GCTGCTCTTGGTCGGAAAGA-3′
TF	5′-CCCAGAAAGCATCACCAAGTG-3′	5′-TGCTCCACAATGATGAGTGTT-3′
β-Actin	5′-TTCAACACCCCAGCCATGT-3′	5′-AGTGGTACGACCAGAGGCATACA-3′

ECE-1, endothelin-converting enzyme-1; ET-1, endothelin-1; ET_A and ET_B, endothelin receptors type A and B; IL-6; interleukin-6; PAI-1, plasminogen activator inhibitor-1; TF, tissue factor.

Hemodynamic Measurements

On day 18, RV pressure-volume loops were determined as previously described (44). After anesthesia with intraperitoneal injection of 25 mg/kg ketamine (Nimatek; Eurovet Animal Health, Bladel, The Netherlands) and 50 mg/kg xylazine (Rompun; Bayer, Leverkusen, Germany), rats were mechanically ventilated via a trachea cannula (Bioflow 20-g intravenous catheter; Vygon, Veenendaal, The Netherlands) with a Minivent type 845 (Hugo Sachs Electronik, March-Hugstetten, Germany; 70% O₂, stroke volume, 270 μl; ventilation rate, 170 strokes/min). After the thorax was opened via the diaphragm, a combined pressure-conductance catheter (model FT212; SciSense, London, ON, Canada) was introduced via the apex into the RV and positioned toward the pulmonary valve. The catheter was connected to a signal processor (model FV898 control box; SciSense), and RV pressures and volumes were recorded digitally and analyzed. After hemodynamic measurements were determined, the heart was removed, fixed in buffered formaldehyde, and processed for histology.

Statistical Analysis

Values are means ± SE. Differences between groups were analyzed by one-way ANOVA for independent samples, followed by Tukey's multiple comparison test, using the GraphPad Prism version 5 software package (San Diego, CA). Differences at P values <0.05 were considered statistically significant.

RESULTS

Effects of Ambrisentan on Growth and Survival

Early concurrent treatment.

On day 10, mean body weight of room air-exposed pups was 18.7 ± 0.9 g (Fig. 2A). Treatment with ambrisentan showed no adverse effects on mean body weight compared with room air controls. After 10 days of hyperoxia exposure, mean body weight was 15.4 ± 0.7 g in the controls and 12.2 ± 0.4 g in the ambrisentan group (P < 0.05). Exposure to hyperoxia resulted in only 40% survival on day 10 vs. 90% after exposure to hyperoxia and treatment with ambrisentan (P < 0.001; Fig. 2B). Room air-exposed pups showed no morbidity or mortality during the first 4 wk after birth. Treatment with l-NAME had no adverse effects on growth and survival in room air- and oxygen-exposed pups compared with controls. Survival and growth were similar in pups treated with ambrisentan and l-NAME compared with pups treated with ambrisentan alone in room air- and oxygen-exposed pups (Ref. 11; data not shown).

Fig. 2.

Growth (A and C) and survival (B and D) on day 10 of prophylactic ambrisentan treatment (n = 12; A and B) and after late treatment and recovery (n = 8; C and D) on days 9 and 18 in RA- and age-matched O₂-exposed pups injected daily with saline or ambrisentan. Data are means ± SE. Open squares and open bar, RA-NaCl; filled squares and hatched bar, RA-ambrisentan; open circles and solid bar, O₂-NaCl; filled circles and shaded bar, O₂-ambrisentan. *P < 0.05; **P < 0.01; ***P < 0.001 vs. age-matched O₂-exposed controls. ΔΔΔP < 0.001 vs. own RA-exposed controls. δδδP < 0.001 vs. own treatment controls on day 9.

Late treatment and recovery.

Mean body weight of room air controls was 17.2 ± 0.3 g on day 9 (Fig. 2C) and 37.4 ± 0.5 g on day 18 and was not influenced by ambrisentan treatment. After 9 days of hyperoxia exposure, mean body weight was 13.1 ± 0.6 g, and this increased to 31.2 ± 1.0 g after 9 days of recovery in room air on day 18. Treatment with ambrisentan decreased body weight compared with oxygen-exposed controls on day 18 (P < 0.01). On days 9 and 18, all room air controls survived (Fig. 2D), but exposure to hyperoxia resulted in 60% survival. Treatment with ambrisentan for 3 days did not affect survival on day 9. All pups that recovered in room air survived until day 18.

Effects of Ambrisentan on Lung Airway Development and Inflammation

Early concurrent treatment.

Compared with room air controls, treatment with ambrisentan, l-NAME, or ambrisentan and l-NAME did not have adverse effects on the number of alveolar crests (Fig. 3I), pulmonary vessel density (Fig. 3, A–D and J), alveolar septal thickness (Fig. 3K), arterial medial wall thickness (Fig. 3L), and influx of macrophages (Fig. 3M) and neutrophilic granulocytes (Fig. 3N), demonstrating normal postnatal alveolar and vascular lung development. A minor effect of ambrisentan was observed on the number of alveolar crests (15% decrease, P < 0.05; Fig. 3I). Oxygen exposure for 10 days (Fig. 3C) resulted in lung edema, a heterogeneous distribution of enlarged air spaces with a decreased number of alveolar crests (2.2-fold, P < 0.001; Fig. 3I), increased alveolar septal thickness (1.9-fold, P < 0.001; Fig. 3K), reduced pulmonary vessel density (2.8-fold, P < 0.001; Fig. 3, C and J), and increased pulmonary arterial medial wall thickness (2.8-fold, P < 0.001; Fig. 3, G and L). Hyperoxia led to a massive inflammatory reaction, characterized by an overwhelming influx of inflammatory cells, including macrophages (6.3-fold, P < 0.001; Fig. 3M) and neutrophils (4.6-fold, P < 0.001; Fig. 3N), compared with room air-exposed controls. Ambrisentan treatment reduced alveolar septal thickness (1.6-fold, P < 0.001; Fig. 3K) and arterial medial wall thickness (1.7-fold, P < 0.001; Fig. 3, H and L) compared with oxygen-exposed controls but had no beneficial effects on the hyperoxia-induced inhibition of alveolarization and angiogenesis and the influx of monocytes and neutrophilic granulocytes. Treatment with l-NAME in room air- and oxygen-exposed pups had no effect on the number of alveolar crests, blood vessel density, septal and arterial medial wall thickness, and the influx of macrophages and neutrophils (Fig. 3, I–N), demonstrating that the beneficial effects of ambrisentan on the hyperoxia-induced increase in septal thickness and arterial medial wall thickness were not affected by l-NAME treatment (Fig. 3, K and L).

Fig. 3.

Lung sections stained for von Willebrand factor (vWF; A–D) or α-smooth muscle actin (ASMA; E–H) and lung morphometry (I–N) of RA (A, B, E, F)- and O₂-exposed pups (C, D, G, H) injected daily with saline (A, C, E, G) or ambrisentan (B, D, F, H) until 10 days of age. Lung morphometry, including the quantifications of alveolar crests (I), number of pulmonary vessels (J), septal thickness (K), arterial medial wall thickness (L), and influx of macrophages (M) and neutrophilic granulocytes (N), was determined on paraffin sections in RA- and O₂-exposed pups injected daily with saline, ambrisentan, N^ω-nitro-l-arginine methyl ester (l-NAME), or ambrisentan + l-NAME. Values are means ± SE (n = 8). a, Alveolus; amb, ambrisentan. Arrows in A–D indicate vWF-positive blood vessels. *P < 0.05; **P < 0.01; ***P < 0.001 vs. age-matched O₂-exposed controls. ΔP < 0.05; ΔΔP < 0.01; ΔΔΔP < 0.001 vs. own RA controls. $$$P < 0.001 vs. ambrisentan-treated O₂-exposed pups.

Late treatment and recovery.

Treatment of room air-exposed pups with ambrisentan showed no adverse effects on alveolar (Fig. 4A) and vascular development (Fig. 4, B and C) on day 18 but showed a minor 1.4-fold increase in arterial medial wall thickness on day 9 compared with room air controls (Fig. 4C). Continuous neonatal exposure to hyperoxia for 9 days resulted in enlarged alveoli, demonstrated by a 2.0-fold decrease in the number of alveolar crests (P < 0.001; Fig. 4A); disturbed vascular development, demonstrated by a 2.2-fold reduction in blood vessel density (P < 0.001; Fig. 4B); and a 2.1-fold increase in arterial medial wall thickness (P < 0.001; Fig. 4C) compared with room air controls. Ambrisentan treatment during the last 3 days of the injurious 9-day hyperoxic period decreased arterial medial wall thickness by 31% (P < 0.001; Fig. 4C) but had no beneficial effects on alveolarization (Fig. 4A) and pulmonary blood vessel density (Fig. 4B). A recovery period of 9 days in room air after hyperoxia-induced lung injury on day 18 had a beneficial effect on the number of alveolar crests (P < 0.001; Fig. 4A) and blood vessel density (P < 0.001; Fig. 4B), but the numbers of alveoli and blood vessels were still reduced after injury and recovery. Treatment with ambrisentan reduced arterial medial wall thickness by 50% (P < 0.001; Fig. 4C) but did not have a beneficial effect on alveolarization (Fig. 4A) and vascularization (Fig. 4B) compared with nontreated experimental BPD pups at the end of the recovery period on day 18.

Fig. 4.

Quantification of alveolar crest (A), number of pulmonary vessels (B), and arterial medial wall thickness (C) determined on paraffin sections after late treatment and recovery on days 9 and 18 in RA-exposed pups injected daily with saline (open bar) or ambrisentan (hatched bar) and O₂-exposed pups injected daily with saline (solid bar) or ambrisentan (shaded bar). Values are means ± SE (n = 8). *P < 0.05: **P < 0.01; ***P < 0.001 vs. age-matched O₂-exposed controls. ΔP < 0.05; ΔΔP < 0.01; ΔΔΔP < 0.001 vs. own RA controls. δδP < 0.01; δδδP < 0.001 vs. own treatment controls on day 9.

Effects of Ambrisentan on Pulmonary Deposition Of Collagen, Elastin, and Fibrin and on Vascular Leakage

Early concurrent treatment.

High levels of collagen III were observed in the perivasculature of large and small blood vessels in normal lung in the absence (Fig. 5A) or presence of ambrisentan (Fig. 5B). Expression in alveolar septa was low or absent. In lungs of pups exposed to hyperoxia for 10 days, collagen III deposition increased 8.9-fold (P < 0.001; Fig. 5I) and was present in thick alveolar septa (Fig. 5C). Treatment with ambrisentan for 10 days reduced collagen III expression by 82% (P < 0.001; Fig. 5I) by preventing hyperoxia-induced extracellular collagen III expression in alveolar septa, but not in the (peri)vascular area (Fig. 5D).

Fig. 5.

Quantification of collagen III (I) and elastin expression (J) on lung paraffin sections of RA (A, B, E, F) and O₂-exposed pups (C, D, G, H) injected daily with saline (A, C, E, G) or ambrisentan (B, D, F, H) until 10 days of age (n = 8), quantification of extravascular fibrin deposition in lung homogenates (n = 10; K), and total protein concentration in bronchoalveolar lavage fluid (BALF; n = 12; L) on day 10 in RA-exposed pups injected daily with saline (open bar) or ambrisentan (hatched bar) and in O₂-exposed pups injected daily with saline (solid bar) or ambrisentan (shaded bar). Values are means ± SE. at, Arteriole. *P < 0.05; **P < 0.01; ***P < 0.001 vs. age-matched O₂-exposed controls. ΔΔΔP < 0.001 vs. own RA controls.

Under normoxia, in the absence (Fig. 5E) or presence of ambrisentan (Fig. 5F), elastin was predominantly present in alveoli on the septal tips and in the wall of blood vessels. Under hyperoxia, elastin expression was decreased 1.4-fold (P < 0.01; Fig. 5J) and was predominantly present in the alveolar walls rather than on septal tips and in the walls of small blood vessels (Fig. 5G). Ambrisentan had no effect on elastin expression in blood vessels and on the hyperoxia-induced irregular elastin expression in alveolar walls in pups with hyperoxia-induced lung injury (Fig. 5, H and J).

Pulmonary fibrin deposition is a sensitive marker for tissue damage in hyperoxia-induced neonatal lung disease and was studied in homogenates as a readout for lung damage (Fig. 5K). Fibrin deposition was at reference levels during normal neonatal pulmonary development on day 10 in the absence or presence of ambrisentan (<15 ng fibrin/mg tissue) and increased 7.9-fold to 65.2 ± 7.9 ng fibrin/mg tissue in lungs of pups exposed to 100% oxygen for 10 days (P < 0.001). Ambrisentan therapy attenuated hyperoxia-induced fibrin deposition by 68% to 21.7 ± 3.6 ng fibrin/mg tissue (P < 0.001).

Total protein concentration in bronchoalveolar lavage fluid (BALF) was determined to investigate the effect of ambrisentan on lung edema by capillary-alveolar leakage (Fig. 5L). The protein concentration on postnatal day 10 increased 9.2-fold after hyperoxia (P < 0.05) but was not affected by treatment with ambrisentan during normoxia or hyperoxia.

mRNA Expression in Lung Tissue

Development of hyperoxia-induced neonatal lung injury.

During normal lung development, mRNA expression of ET-1 had increased (2-fold on day 6, P < 0.01; Fig. 6A), and expression of ET_B was higher on days 3 (1.8-fold, P < 0.001), 6 (1.7-fold, P < 0.01), and 10 (1.5-fold, P < 0.01; Fig. 6D) than on day 1. mRNA expression in adult lung of ET-1 (2.9-fold, P < 0.001) and ECE-1 (1.7-fold, P < 0.001) in adult lung was higher than on day 10. Exposure to hyperoxia resulted in an increase in expression of ET-1 (3.3-fold and 6.5-fold on days 6 and 10, respectively) and a decrease in expression on day 10 for ET_A (1.9-fold, P < 0.001; Fig. 6C) and ET_B (1.8-fold, P < 0.001) compared with age-matched room air controls. ECE-1 was not differentially expressed during normal neonatal development or after exposure to hyperoxia, but expression increased toward adulthood (Fig. 6B).

Fig. 6.

Relative mRNA expression in lung homogenates of endothelin-1 (A), endothelin-converting enzyme 1 (ECE-1; B), endothelin receptor type A (ET_A; C), and endothelin receptor type B (ET_B; D) on days 1, 3, 6, and 10 after birth in RA (open bar) and O₂-exposed pups (solid bar). pd, Postnatal day. Values are means ± SE (n = 8). ***P < 0.001 vs. age-matched O₂-exposed controls. ΔΔP < 0.01; ΔΔΔP < 0.001 vs. neonatal day 1. $$$P < 0.001 vs. RA control on day 10.

Early concurrent treatment.

Treatment with ambrisentan for 10 days in room air resulted in a minor decrease in mRNA expression of the proinflammatory factor interleukin (IL)-6 (Fig. 7A) and the procoagulant protein tissue factor (TF; Fig. 7B) and an increase in the mRNA expression of the antifibrinolytic factor plasminogen activator inhibitor-1 (PAI-1; Fig. 7C), the growth factor amphiregulin (Fig. 7D), ET-1 (Fig. 7E), and its converting enzyme ECE-1 (Fig. 7F). Expression of the endothelin receptors ET_A and ET_B did not change during normoxia. Ten days of oxygen exposure resulted in a 12-fold increase in mRNA expression of IL-6 (P < 0.001), a 2.1-fold increase in TF (P < 0.001), a 41-fold increase in PAI-1 (P < 0.001), a 5.3-fold increase in amphiregulin (P < 0.001), and a 3.2-fold increase in ET-1 (P < 0.001), whereas mRNA expression of ET_A decreased 1.5-fold (P < 0.001) and that of ET_B decreased 2.1-fold (P < 0.001) in lungs of oxygen-exposed pups compared with room air-exposed controls. Treatment of oxygen-exposed pups with ambrisentan for 10 days resulted in a 21% reduction of the mRNA expression of TF (P < 0.01) and a 52% reduction in amphiregulin mRNA (P < 0.01) vs. a 79% increase in mRNA expression of ET-1 (P < 0.001) and a 25% increase in ET_B mRNA (P < 0.05) compared with oxygen-exposed pups.

Fig. 7.

Relative mRNA expression in lung homogenates after early concurrent treatment of interleukin-6 (IL-6; A) tissue factor (TF; B), plasminogen activator inhibitor type 1 (PAI-1; C), amphiregulin (D), endothelin-1 (E), ECE-1 (F), ET_A (G), and ET_B (H) on day 10 in RA-exposed pups injected daily with saline (open bar) or ambrisentan (hatched bar) and O₂-exposed pups injected daily with saline (solid bar) or ambrisentan (shaded bar). Values are means ± SE (n = 10). *P < 0.05; **P < 0.01; ***P < 0.001 vs. age-matched O₂-exposed controls. ΔP < 0.05; ΔΔP < 0.01; ΔΔΔP < 0.001 vs. RA controls.

Heart Development and RVH

Early concurrent treatment.

Treatment with ambrisentan and/or l-NAME for 10 days during normal neonatal development had no effect on cardiac hypertrophy (Fig. 8, A–C, and Table 2). Exposure to hyperoxia for 10 days resulted in RVH as demonstrated by a 1.6-fold increase in the ratio RV/LV free wall thickness (Fig. 8, A and B) and a 1.5-fold increase in weight ratio RV/(LV + IVS) (Fig. 8C) compared with room air controls (P < 0.001). Treatment with ambrisentan resulted in a significant regression of RVH as demonstrated by a decrease in weight ratio and relative RV free wall thickness by 21% (P < 0.01) and 40% (P < 0.001), respectively, compared with the oxygen-exposed controls. The beneficial effects of ambrisentan on RV free wall thickness were not affected by l-NAME treatment (Fig. 8B).

Fig. 8.

A: paraffin heart sections stained with hematoxylin and eosin after early concurrent treatment in room air-exposed controls treated with saline (RA) or ambrisentan (RA-amb) and age-matched O₂-exposed controls injected daily with either saline (O₂) or ambrisentan (O₂-amb). B: RVH depicted as RV/LV wall thickness ratio on day 10 after early concurrent treatment (n = 8, B) in RA- and O₂-exposed pups injected daily with saline, ambrisentan, l-NAME, or ambrisentan + l-NAME. C–E: RVH depicted as the weight ratio RV/(LV + IVS) (n = 12; C) and as RV/LV wall thickness ratio after late treatment and recovery (n = 8; D) on days 9 and 18, and RV function depicted as the peak RV pressure on day 18 after injury and recovery (n = 8; E) in RA-exposed pups injected daily with saline (open bar) or ambrisentan (hatched bar) and O₂-exposed pups injected daily with saline (solid bar) or ambrisentan (shaded bar). IVS, interventricular septum. Data are means ± SE. Cardiac characteristics are presented in Tables 2 and 3. *P < 0.05; **P < 0.01; ***P < 0.001 vs. age-matched O₂-exposed controls. ΔP < 0.05; ΔΔΔP < 0.001 vs. RA controls. $$$P < 0.001 vs. ambrisentan-treated O₂-exposed pups.

Table 2.

Cardiac characteristics in early concurrent treatment

		RA		O2
	Day	Saline	Ambrisentan	Saline	Ambrisentan
RV free wall thickness, μm	10	277 ± 17†	203 ± 9†	446 ± 23	261 ± 21†
LV free wall thickness, μm	10	870 ± 24	705 ± 21†‡	901 ± 29	878 ± 45
IVS thickness, μm	10	801 ± 19	666 ± 10‡	740 ± 25	659 ± 21
RV/LV ratio	10	0.32 ± 0.02†	0.29 ± 0.01†	0.50 ± 0.02	0.30 ± 0.03†
RV weight, mg	10	22.6 ± 1.1	22.4 ± 1.3†	25.2 ± 0.9	18.0 ± 0.8‡
LV + IVS weight, mg	10	85.8 ± 2.1†	85.3 ± 2.5†	67.8 ± 2.3	60.8 ± 2.7§
RV/(LV + IVS) ratio	10	0.27 ± 0.02†	0.26 ± 0.02†	0.38 ± 0.02	0.30 ± 0.01*

Values are means ± SE in room air (RA)- and oxygen (O₂)-exposed rat pups treated with saline (controls) or ambrisentan.

RV, right ventricular; LV, left ventricular; IVS, interventricular septum.

^*P < 0.01;

^†P < 0.001 vs. age-matched O₂-exposed controls.

^‡P < 0.01;

^§P < 0.001 vs. RA-exposed controls.

Late treatment and recovery.

Nine days of hyperoxic lung injury resulted in a 1.5-fold increase in RV/LV free wall thickness compared with that of room air controls (P < 0.001; Fig. 8D and Table 3), which was attenuated after 3 days of ambrisentan treatment on day 9 (33%; P < 0.001). A recovery period of 9 days did not reduce RVH in the nontreated pups, but on day 18 the RV/LV free wall thickness ratio in the ambrisentan group was smaller (18%; P < 0.05; Fig. 8D).

Table 3.

Cardiac characteristics in late treatment and recovery

		RA		O2
	Day	Saline	Ambrisentan	Saline	Ambrisentan
RV free wall thickness, μm	9	212 ± 48‡	214 ± 32‡	352 ± 24	262 ± 14†
	18	268 ± 9‡	305 ± 20‡	417 ± 17	369 ± 18§
LV free wall thickness, μm	9	672 ± 26	718 ± 23	740 ± 35	829 ± 27§
	18	948 ± 19	1037 ± 53	962 ± 32	1028 ± 36
IVS thickness, μm	9	576 ± 30	561 ± 18	605 ± 32	609 ± 21
	18	857 ± 12	884 ± 79	805 ± 32	819 ± 30
RV/LV ratio	9	0.31 ± 0.02‡	0.30 ± 0.02‡	0.48 ± 0.03	0.32 ± 0.02‡
	18	0.28 ± 0.02‡	0.29 ± 0.02‡	0.44 ± 0.02	0.36 ± 0.02*

Values are means ± SE in RA- and O₂-exposed rat pups treated with saline (controls) or ambrisentan.

^*P < 0.05;

^†P < 0.01;

^‡P < 0.001 vs. age-matched O₂-exposed controls.

^§P < 0.01 vs. RA-exposed controls.

Right Ventricular Function

Late treatment and injury recovery model.

After 9 days of hyperoxia and 9 days of recovery in room air, peak RV pressure was higher (P < 0.001) than in room air controls, leading to hyperoxia-induced PAH (Fig. 8E). Treatment with ambrisentan reduced hyperoxia-induced peak RV pressure by 31% (P < 0.001) compared with oxygen-exposed controls, i.e., ambrisentan attenuates hyperoxia-induced PAH on day 18.

DISCUSSION

Prophylactic treatment with ambrisentan in neonatal rat pups exposed to prolonged hyperoxia, an in vivo model for experimental CLD (46), improved survival by attenuating lung injury and RVH. The beneficial effects of the specific ET_A antagonist ambrisentan on cardiopulmonary injury included a reduction in pulmonary extravascular fibrin deposition, collagen III expression, tissue factor mRNA expression, alveolar septal thickness, arterial medial wall thickness, and thickness of the RV free wall. These effects were not affected by l-NAME administration. Ambrisentan treatment had no beneficial effects on lung vascularization, inflammation (pulmonary influx of macrophages and neutrophils, and IL-6 mRNA expression), and capillary alveolar leakage (total protein content in BALF). In the injury recovery model, treatment with ambrisentan attenuated PAH, RVH, and RV peak pressure but did not attenuate impaired alveolar or vascular development in neonatal rats with CLD, demonstrating that PAH-induced RVH is still reversible in the neonatal period (present study and Refs. 10 and 11).

Treatment of neonatal hyperoxic lung disease with ambrisentan resulted in a 20% reduction in body weight on day 10 in the prophylactic model and a small but significant 10% reduction in the injury recovery model on day 18. Side effects of the endothelin receptor antagonist include fluid retention, liver problems, birth defects, anemia, nausea, and loss of appetite (4). Liver toxicity is the most concerning side effect of ET receptor antagonists, but ambrisentan is less hepatotoxic than bosentan in clinical studies (4). Nausea and loss of appetite may directly contribute to the observed weight loss after treatment with ambrisentan. Furthermore, the role of ambrisentan as an ergonic is under current clinical investigation. Although treatment with ambrisentan resulted in a reduction in body weight, gross abnormalities in the abdominal and thoracic organs were not seen. However, it is possible that microscopic or biochemical metabolic abnormalities may have been induced by the relatively high doses of ambrisentan used in this model. l-NAME treatment in room air- and oxygen-exposed pups had no adverse effects on lung and heart parameters. These findings demonstrate that endothelin receptor antagonists may be suitable for therapeutic intervention in preterm infants with severe CLD and PAH.

Ambrisentan treatment of experimental BPD reduces extravascular pulmonary fibrin deposition and mRNA expression of tissue factor, the physiological initiator of coagulation. Extravascular fibrin deposition suggests capillary alveolar leakage of plasma proteins, including fibrinogen leakage into the alveolar lumen followed by local conversion into fibrin by thrombin. Ambrisentan did not affect hyperoxia-induced capillary alveolar leakage of plasma proteins and the differential mRNA expression of PAI-1, the key regulator of fibrinolysis. This suggests that ambrisentan may reduce initiation of coagulation rather than stimulation of fibrin degradation or reduction of capillary alveolar leakage in rats with CLD.

ET-1 and both endothelin receptors ET_A and ET_B are expressed in the lung in multiple cell types, including bronchial and alveolar epithelium, endothelium, vascular smooth muscle cells, and inflammatory cells (present study and Refs. 20, 28, 35, and 39). The endothelin receptors ET_A and ET_B and their ligand ET-1 are differentially expressed at the mRNA level in hyperoxia-induced lung injury with an increased expression of ET-1, confirmed at the protein level, and a decreased expression of both receptors. This adaptive response toward hyperoxia suggests a role for endothelin/endothelin receptor signaling in the pathophysiology of severe experimental BPD, in which arrested alveolarization and pulmonary hypertension play a pivotal role. In these experiments we chose a specific ET_A receptor antagonist to inhibit the adverse effects of endothelin receptor binding on vascular smooth muscle cells, i.e., PAH by vasoconstriction (14), and to preserve the beneficial effects of ET_B-mediated signaling in endothelial cells, i.e., pulmonary endothelin reuptake from the circulation and prostacyclin- and NO-mediated vasodilation (12). We demonstrated previously that activation of the eNOS/cGMP pathway by apelin or sildenafil (10, 11) induces alveolarization, angiogenesis, and vasodilation and reduces inflammation and coagulation in experimental BPD. In this study we only observed beneficial effects of ambrisentan treatment of hyperoxia-induced neonatal lung disease on alveolar septal thickness and arterial medial wall thickness, which were independent of NOS activation, and on fibrin deposition, but not on alveolar and vascular development. Our findings in ambrisentan-treated neonatal rats with hyperoxia-induced lung injury confirm data obtained in neonatal hypoxia-induced lung injury in mice, in which ET_A blocking does not improve inhibition of alveolar development (32), but are in sharp contrast with the improved alveolarization and reduced inflammation seen in adult rats with cigarette smoke extract-induced lung emphysema treated prophylactically with endothelin receptor ET_A (BQ-123) or ET_A/ET_B antagonists (bosentan) (7). Alveolar enlargement is caused in (preterm) infants by an arrest in alveolar development and in adults by alveolar destruction (5, 22). This difference in causation of emphysema may explain the differential response of endothelin receptor antagonists in infants and adults.

Although lung tissue damage was attenuated by prophylactic ambrisentan treatment, as shown by a reduction in alveolar septal thickness and collagen III and extravascular fibrin deposition, ET receptor antagonists did not dampen the inflammatory response significantly (a tendency toward lower IL-6 mRNA levels and the influx of macrophages and neutrophils was observed in lungs of ambrisentan-treated oxygen-exposed pups compared with saline-treated controls). This observation is again in contrast with the findings in adult animals. In rat models of cigarette smoke extract-induced lung emphysema and bleomycin-induced pulmonary fibrosis and in pigs with orthotopic liver transplantation treatment with ET receptor antagonists, reduced lung tissue damage was associated with an anti-inflammatory response (7, 33, 43). This discrepancy may be due to differences in maturation with different triggers for lung injury.

In the mammalian lung, elastin plays an important role in lung development as demonstrated in elastin-deficient mice, with defective branching of the terminal airways and associated vascular development (48). In rat pups with experimental CLD, reduced alveolarization and vascularization was associated with reduced elastin expression and a redistribution of elastic fibers from the septal tips to the alveolar walls. These changes in pulmonary elastin expression are similar in multiple animal models of neonatal chronic lung disease, such as ventilator-induced lung injury in neonatal mice (29) and preterm lambs (6), hyperoxic lung injury in preterm rabbits (27), and premature infants who have died from BPD (26, 41). Because elastin is expressed at high levels in blood vessel walls, reduced elastin expression in the lung under hyperoxia can at least in part be explained by a reduction in pulmonary vascularization.

The beneficial effects of ambrisentan treatment are mainly mediated via prevention and reversal of PAH-induced RVH in the absence of an anti-inflammatory response. The results argue against a significant role of ambrisentan in inflammation-mediated vasoconstriction and suggest that ambrisentan has a direct effect on vascular smooth cell proliferation and/or contraction via effective ET_A blockage under conditions with increased ET-1 expression caused by tissue damage and inflammation. Prevention and reversal of PAH-induced RVH by ambrisentan in hyperoxia-induced neonatal lung disease is in agreement with observations in multiple adult rodent models, such as monocrotaline-, hypoxia- and bleomycin-induced PAH, in which treatment with ET receptor antagonists can prevent and/or reverse PAH-induced RVH (3, 36, 50). Extrapolation of our results in hyperoxia-exposed neonatal rats to preterm infants with respiratory failure raises the expectation of a beneficial effect of ambrisentan on both PH and RVH even with ongoing lung inflammation, which are the major reasons for mortality or severe morbidity in preterm infants with severe CLD.

GRANTS

This study was supported by National Institutes of Health Grants 1R01 HL092158 and 1R01 ES015330 (F. J. Walther).

DISCLOSURES

No conflicts of interest, financial or otherwise are declared by the authors.

AUTHOR CONTRIBUTIONS

G.T.W., Y.P.d.V., and F.J.W. conception and design of research; G.T.W., E.H.L., Y.P.d.V., R.M.S., P.S., and H.J.B. performed experiments; G.T.W., E.H.L., Y.P.d.V., R.M.S., P.S., and H.J.B. analyzed data; G.T.W., E.H.L., Y.P.d.V., P.S., H.J.B., and F.J.W. interpreted results of experiments; G.T.W., E.H.L., Y.P.d.V., R.M.S., and P.S. prepared figures; G.T.W. drafted manuscript; G.T.W., H.J.B., and F.J.W. edited and revised manuscript; G.T.W., E.H.L., Y.P.d.V., P.S., H.J.B., and F.J.W. approved final version of manuscript.