Apelin attenuates hyperoxic lung and heart injury in neonatal rats

Abstract

Rationale: Apelin, a potent vasodilator and angiogenic factor, may be a novel therapeutic agent in neonatal chronic lung disease, including bronchopulmonary dysplasia.

Objectives: To determine the beneficial effect of apelin in neonatal rats with hyperoxia-induced lung injury, a model for premature infants with bronchopulmonary dysplasia.

Methods: The cardiopulmonary effects of apelin treatment (62 μg/kg/d) were studied in neonatal rats by exposure to 100% oxygen, using two treatment strategies: early concurrent treatment during continuous exposure to hyperoxia for 10 days and late treatment and recovery in which treatment was started on Day 6 after hyperoxic injury for 9 days and continued during the 9-day recovery period. We investigated in both models the role of the nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in apelin treatment by specific inhibition of the nitric oxide synthase activity with N(ω)-nitro-L-arginine methyl ester (L-NAME, 25 mg/kg/d).

Measurements and main results: Parameters investigated include survival, lung and heart histopathology, pulmonary fibrin deposition and inflammation, alveolar vascular leakage, lung cGMP levels, right ventricular hypertrophy, and differential mRNA expression in lung and heart tissue. Prophylactic treatment with apelin improved alveolarization and angiogenesis, increased lung cGMP levels, and reduced pulmonary fibrin deposition, inflammation, septum thickness, arteriolar wall thickness, and right ventricular hypertrophy. These beneficial effects were completely absent in the presence of L-NAME. In the injury-recovery model apelin also improved alveolarization and angiogenesis, reduced arteriolar wall thickness, and attenuated right ventricular hypertrophy.

Conclusions: Apelin reduces pulmonary inflammation, fibrin deposition, and right ventricular hypertrophy, and partially restores alveolarization in rat pups with neonatal hyperoxic lung injury via a nitric oxide synthase-dependent mechanism.

Figure 1.

Lung sections stained immunohistochemically for (A–C) apelin or (D–F) APJ after early concurrent treatment on Day 10 in (A, D) room air and age-matched O₂-exposed pups daily injected with (B, E) saline or (C, F) apelin. (G) APJ mRNA expression by in situ hybridization in a room air–exposed pup on Day 10. Arrows in G indicate APJ mRNA expression in alveolar capillaries and epithelial type 2 cells. a = alveolus; b = bronchus; bv = blood vessel.

Figure 2.

(A, D) Growth and(B, C, E) survival at Day 10 after early concurrent treatment (n = 12, shaded bars; A, B) and after late treatment and recovery (n = 8; D, E) on Days 9 (open bars) and 18 (solid bars) in room air (RA) and age-matched O₂-exposed pups (O₂) daily injected with either saline, apelin, N_ω-nitro-l-arginine methyl ester (l-NAME), or a combination of apelin and l-NAME. Kaplan-Meier survival curve of apelin-treated rat pups (solid circles), age-matched O₂-exposed control pups (open triangles) and RA-exposed control pups (open squares) during the first 14 days after birth (n = 12; C). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus age-matched O₂-exposed control pups. ^ΔP < 0.05, ^ΔΔP < 0.01, ^ΔΔΔP < 0.001 versus own room air–exposed control pups. ^$P < 0.05 and ^$$$P < 0.001 versus apelin-treated O₂ pups. ^δδδP < 0.001 versus own treatment control pups in hyperoxic period. nd = not determined.

Figure 3.

Lung sections stained for von Willebrand Factor (vWF; A–H) and lung morphometry (I–P) of room air– (RA; A–D) and O₂-exposed pups (O₂; E–H) daily injected with either saline (A, E), apelin (B, F), N_ω-nitro-l-arginine methyl ester (l-NAME; C, G) or a combination of apelin and l-NAME (D, H) at 10 days of age after early concurrent treatment. Lung morphometry, including the quantification of mean linear intercept (I), alveolar crest (per field in J and per tissue ratio in K), number of pulmonary vessels (L), septum thickness (M), medial wall thickness (N) and influx of macrophages (O) and neutrophilic granulocytes (P) was determined on paraffin sections in RA and O₂ pups daily injected either with saline, apelin, and/or l-NAME. Values are expressed as mean ± SEM (n = 8). Arrows in A–H indicate vWF-positive blood vessels. **P < 0.01 and ***P < 0.001 versus age-matched O₂-exposed control pups. ^ΔP < 0.05 and ^ΔΔΔP < 0.001 versus own RA control pups. ^$P < 0.05, ^$$P < 0.01, ^$$$P < 0.001 versus apelin-treated O₂ pups. a = alveolus.

Figure 4.

Quantification of (A) mean linear intercept, (B) alveolar crest, (C) number of pulmonary vessels, and (D) medial wall thickness determined on paraffin sections after late treatment and recovery on Days 9 (open bars) and 18 (solid bars) in room air (RA) and O₂-exposed pups (O₂) daily injected either with saline, apelin, N_ω-nitro-l-arginine methyl ester (l-NAME), or a combination of apelin and l-NAME. Values are expressed as mean ± SEM (n = 8). ***P < 0.001 versus age-matched O₂-exposed control pups. ^ΔΔΔP < 0.001 versus own RA control pups. ^$P < 0.05, ^$$P < 0.01, ^$$$P < 0.001 versus apelin-treated O₂ pups. ^δP < 0.05, ^δδP < 0.01, ^δδδP < 0.001 versus own treatment control pups in hyperoxic period. nd = not determined.

Figure 5.

Quantification of (A) fibrin deposition in lung homogenates, (B) total protein concentration in bronchoalveolar lavage fluid (BALF), and (C–E) expression of β-fibrin on lung sections after early concurrent treatment on Day 10 in room air (RA) and O₂-exposed pups (O₂) daily injected either with saline, apelin, l-NAME, or a combination of apelin and N_ω-nitro-l-arginine methyl ester (l-NAME). Values are expressed as mean ± SEM (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001 versus age-matched O₂-exposed control pups. ^ΔΔΔP < 0.001 versus own RA control pups. ^$$$P < 0.001 versus apelin-treated O₂ pups. a = alveolus; b = bronchus.

Figure 6.

Quantification of cyclic guanosine monophosphate (cGMP) in lung homogenates after early concurrent treatment on Day 10 in room air–exposed (RA) and O₂-exposed pups (O₂) daily injected either with saline, apelin, N_ω-nitro-l-arginine methyl ester (l-NAME), or a combination of apelin and l-NAME. Values are expressed as mean ± SEM (n = 10). *P < 0.05, ***P < 0.001 versus age-matched O₂-exposed control pups. ^ΔP < 0.05, ^ΔΔΔP < 0.001 versus own room air-exposed control pups. ^$$$P < 0.001 versus apelin-treated O₂ pups.

Figure 7.

Relative mRNA expression in lung homogenates after early concurrent treatment of (A) tissue factor (TF), (B) plasminogen activator inhibitor type 1 (PAI-1), (C) amphiregulin, (D) vascular endothelial growth factor (VEGFA), (E) vascular endothelial growth factor receptor type 2 (VEGFR2), (F) apelin (F), (G) APJ, (H) PDE5, and (I) endothelial nitric oxide synthase (eNOS) on Day 10 in room air–exposed (RA) and O₂-exposed pups (O₂) daily injected either with saline, apelin, N_ω-nitro-l-arginine methyl ester (l-NAME), or a combination of apelin and l-NAME. Values are expressed as mean ± SEM (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001 versus age-matched O₂-exposed control pups. ^ΔP < 0.05, ^ΔΔP < 0.01, ^ΔΔΔP < 0.001 versus own RA control pups. ^$P < 0.05, ^$$P < 0.01, ^$$$P < 0.001 versus apelin-treated O₂ pups.

Figure 8.

Right ventricular hypertrophy depicted as (A) the weight ratio RV/(LV+IVS) at Day 10 after early concurrent treatment (n = 10, shaded bars) and (B) as RV/LV wall thickness ratio after late treatment and recovery (n = 8) on Days 9 (open bars) and 18 (solid bars) in room air–exposed control pups (RA) and age-matched O₂-exposed control pups (O₂) daily injected with either saline, apelin, N_ω-nitro-l-arginine methyl ester (l-NAME), or a combination of apelin and l-NAME. Data are expressed as mean ± SEM. Cardiac characteristics are presented in Table E3. **P < 0.01, ***P < 0.001 versus age-matched O₂-exposed control pups. ^ΔΔΔP < 0.001 versus room air–exposed control pups. ^$$$P < 0.001 versus apelin-treated O₂ pups. Paraffin heart sections stained with hematoxylin and eosin after late treatment and recovery on Days 9 (C) and 18 (D) in room air–exposed control pups (RA) and age-matched O₂-exposed control pups (O₂) daily injected with either saline or apelin. IVS = interventricular septum; LV = left ventricle; nd = not determined; RV = right ventricle.

Figure 9.

Relative mRNA expression in (A–C) the right ventricle (RV) and (D–F) left ventricle and interventricular septum (LV+S) after early concurrent treatment of (A, D) apelin, (B, E) APJ, and (C, F) brain natriuretic peptide (BNP) in room air–exposed control pups (RA) and age-matched O₂-exposed control pups (O₂) daily injected with either saline, apelin, N_ω-nitro-l-arginine methyl ester (l-NAME), or a combination of apelin and l-NAME. Data are expressed as mean ± SEM (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001 versus age-matched O₂-exposed control pups. ^ΔΔP < 0.01, ^ΔΔΔP < 0.001 versus own room air–exposed control pups. ^$P < 0.05, ^$$P < 0.01, ^$$$P < 0.001 versus apelin-treated O₂ pups.