Agonists of MAS oncogene and angiotensin II type 2 receptors attenuate cardiopulmonary disease in rats with neonatal hyperoxia-induced lung injury

Abstract

Stimulation of MAS oncogene receptor (MAS) or angiotensin (Ang) receptor type 2 (AT2) may be novel therapeutic options for neonatal chronic lung disease (CLD) by counterbalancing the adverse effects of the potent vasoconstrictor angiotensin II, consisting of arterial hypertension (PAH)-induced right ventricular hypertrophy (RVH) and pulmonary inflammation. We determined the cardiopulmonary effects in neonatal rats with CLD of daily treatment during continuous exposure to 100% oxygen for 10 days with specific ligands for MAS [cyclic Ang-(1-7); 10-50 μg·kg(-1)·day(-1)] and AT2 [dKcAng-(1-7); 5-20 μg·kg(-1)·day(-1)]. Parameters investigated included lung and heart histopathology, fibrin deposition, vascular leakage, and differential mRNA expression in the lungs of key genes involved in the renin-angiotensin system, inflammation, coagulation, and alveolar development. We investigated the role of nitric oxide synthase inhibition with N(ω)-nitro-l-arginine methyl ester (25 mg·kg(-1)·day(-1)) during AT2 agonist treatment. Prophylactic treatment with agonists for MAS or AT2 for 10 days diminished cardiopulmonary injury by reducing alveolar septum thickness and medial wall thickness of small arterioles and preventing RVH. Both agonists attenuated the pulmonary influx of inflammatory cells, including macrophages (via AT2) and neutrophils (via MAS) but did not reduce alveolar enlargement and vascular alveolar leakage. The AT2 agonist attenuated hyperoxia-induced fibrin deposition. In conclusion, stimulation of MAS or AT2 attenuates cardiopulmonary injury by reducing pulmonary inflammation and preventing PAH-induced RVH but does not affect alveolar and vascular development in neonatal rats with experimental CLD. The beneficial effects of AT2 activation on experimental CLD were mediated via a NOS-independent mechanism.

Keywords: angiotensin-(1-7); bronchopulmonary dysplasia; lung inflammation; pulmonary hypertension; right ventricular hypertrophy.

Fig. 1.

Pilot experiment to find the optimal dose of MAS agonist [cAng-(1-7); A] or angiotensin (Ang) receptor type 2 (AT2) agonist [dKcAng-(1-7); B] for treatment of experimental chronic lung disease (CLD) by determining right ventricular hypertrophy depicted as right to left free ventricular wall thickness (RV/LV) ratio on day 10 after treatment (N = 6) in room air (RA), pups injected daily with saline (open bar) and O₂-exposed pups (O₂) injected daily with saline (solid bar) or agonist (shaded bars): cAng-(1-7) (5 and 15 μg/kg twice a day) and dKcAng-(1-7) (2.5, 5, and 10 μg/kg twice a day). Data are expressed as means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. age-matched O₂-exposed controls.

Fig. 2.

Growth (A) and survival (B) on day 10 (N = 12) in RA- and age-matched O₂-exposed pups injected daily with saline or agonist for MAS [cAng-(1-7), 5 μg/kg twice a day] or AT2 [dKcAng-(1-7), 5 μg/kg twice a day]. Data are expressed as means ± SE. Open bar, RA-NaCl; hatched bar, RA-agonist; solid bar, O₂-NaCl; shaded bar, O₂-agonist. Data are expressed as means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. age-matched O₂-exposed controls. ΔΔΔP < 0.001 vs. own RA-exposed controls.

Fig. 3.

Representative lung sections stained for von Willebrand factor (vWF; A–D), α-smooth muscle actin (ASMA; E–H), the monocyte and macrophage marker ED1 (I–L), or myeloperoxidase (MPO) as a marker for neutrophilic granulocytes (M–P) of RA (A, E, I, and M) and O₂-exposed pups (O₂; B–D, F–H, J–L, and N–P) injected daily with saline (A, B, E, F, I, J, M, and N), MAS agonist [cAng-(1-7), 5 μg/kg twice a day; C, G, K, and O] agonist [dKcAng-(1-7), 5 μg/kg twice a day; D, H, L, and P] until 10 days of age. Values are expressed as means ± SE (N = 8). a = alveolus. Arrows in A–D indicate vWF-positive blood vessels.

Fig. 4.

Lung morphometry, including the quantifications of alveolar crests (A), number of pulmonary vessels (B), septal thickness (C), arterial medial wall thickness (D), and influx of macrophages (E) and neutrophilic granulocytes (F), was determined on paraffin sections in RA pups injected daily with saline (open bar) or agonist (hatched bar) and O₂ pups injected daily with saline (solid bar) or agonist (shaded bar): 5 μg/kg twice a day of MAS agonist [cAng-(1-7)] or AT2 agonist [dKcAng-(1-7)] until 10 days of age. Values are expressed as means ± SE (N = 8). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. age-matched O₂-exposed controls. ΔP < 0.05, ΔΔP < 0.01, and ΔΔΔP < 0.001 vs. own RA controls.

Fig. 5.

Lung morphometry, including the quantifications of alveolar crests (A), number of pulmonary vessels (B), septal thickness (C), arterial medial wall thickness (D), and influx of macrophages (E) and neutrophilic granulocytes (F) was determined on paraffin sections in RA- and O₂-exposed pups injected daily with 100 μl saline, AT2 agonist [LP2–3: dKcAng-(1-7); 5 μg/kg twice a day], N^ω-nitro-l-arginine methyl ester (l-NAME; 25 mg·kg⁻¹·day⁻¹), or LP2–3 and l-NAME (shaded bars). Values are expressed as means ± SE (N = 8). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. age-matched O₂-exposed controls. ΔP < 0.05, ΔΔP < 0.01, and ΔΔΔP < 0.001 vs. own RA controls. $$$P < 0.001 vs. AT2 agonist-treated O₂ pups.

Fig. 6.

Quantification of extravascular fibrin deposition in lung homogenates (N = 10, A) and total protein concentration in bronchoalveolar lavage fluid (BALF; N = 12, B) as a marker for lung injury on day 10 in RA-exposed pups injected daily with saline (open bar), MAS agonist [cAng-(1-7), 5 μg/kg twice a day; hatched bar] or AT2 agonist [dKcAng-(1-7), 5 μg/kg twice a day; hatched bar] and in O₂-exposed pups (O₂) injected daily with saline (solid bar) or agonist (shaded bar). Values are expressed as means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. age-matched O₂-exposed controls. ΔΔΔP < 0.001 vs. own RA controls.

Fig. 7.

Relative mRNA expression of angiotensin receptor type 1a (AT1a; A), AT2 (B), MAS (C), angiotensinogen (D), angiotensin converting enzyme 1 (ACE-1; E) and ACE-2 (F) in pups on days 1, 3, 6, and 10 after birth and in adults during normal development (open bars) and after exposure to 100% oxygen (solid bars). Values are expressed as means ± SE (N = 8). **P < 0.01, and ***P < 0.001 vs. own age-matched O₂-exposed controls. ΔP < 0.05, ΔΔP < 0.01, and ΔΔΔP < 0.001 vs. RA control on day 1. $P < 0.05, $$P < 0.01, and $$$P < 0.001 vs. RA control on day 10.

Fig. 8.

Relative mRNA expression in lung homogenates after early concurrent treatment of monocyte chemoattractant protein 1 (MCP1; A), chemokine-induced neutrophilic chemoattractant-1 (CINC1; B), tissue factor (TF; C), AT1a (D), AT2 (E), MAS (F), angiotensinogen (G), angiotensin converting enzyme 1 (ACE-1; H), angiotensin converting enzyme 2 (ACE-2; I) on day 10 in RA pups injected daily with saline (open bar) or MAS agonist [cAng-(1-7), 5 μg/kg twice a day; hatched bar] or AT2 agonist [dKcAng-(1-7), 5 μg/kg twice a day; hatched bar] and O₂-exposed pups injected daily with saline (solid bar) or agonist (shaded bar). Values are expressed as means ± SE (N = 8). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. age-matched O₂-exposed controls. ΔΔΔP < 0.001 vs. RA controls.

Fig. 9.

Right ventricular (RV) hypertrophy (RVH) was determined by morphometry in paraffin sections stained with hematoxylin and eosin (A) and depicted as RV-to left ventricular (LV) wall thickness ratio (RV/LV; B and D) or by determining the weight ratio RV/(LV+IVS), where IVS is interventricular septum (C) on day 10 after daily treatment in RA pups injected daily with saline (open bar), MAS agonist [cAng-(1-7), 5 μg/kg twice a day; hatched bar (B)], or AT2 agonist [dKcAng-(1-7), 5 μg/kg twice a day; hatched bar; B and C] and O₂-exposed pups injected daily with saline (solid bar) or agonist (shaded bar). Effect of l-NAME (25 mg·kg⁻¹·day⁻¹) on AT2 agonist treatment of hyperoxia-induced RVH by morphometry in paraffin sections stained with hematoxylin and eosin and depicted as RV/LV wall thickness ratio (D). Data are expressed as means ± SE (N = 8). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. own age-matched O₂-exposed controls. ΔP < 0.05 and ΔΔΔP < 0.001 vs. RA control on day 1. $P < 0.05 vs. AT2 agonist-treated O₂ pups.