Volume 147, Issue 4, Pages 969-978 (April 2015) The Apelin-APJ Axis Is an Endogenous Counterinjury Mechanism in Experimental Acute Lung Injury  Xiao-Fang Fan, MD, Feng Xue, MD, Yue-Qi Zhang, MD, Xue-Ping Xing, MD, Hui Liu, MD, Sun-Zhong Mao, MD, PhD, Xiao-Xia Kong, MD, Yu-Qi Gao, MD, PhD, Shu F. Liu, MD, PhD, Yong-Sheng Gong, MD, PhD  CHEST  Volume 147, Issue 4, Pages 969-978 (April 2015) DOI: 10.1378/chest.14-1426 Copyright © 2015 The American College of Chest Physicians Terms and Conditions

Figure 1 ARDS is associated with increased apelin level. Rats in the Con or ARDS group were injected with saline or oleic acid (OA) (0.2 mL/kg, IV). At 6 h after OA injection, plasma, lung tissue, and BALF levels of apelin-36 and apelin-12/13 were measured. A and D, Plasma. B and E, Lung tissue. C and F, BALF. Data are presented as mean ± SD of 10 rats in each group.*P <.05 compared with Con. BALF = BAL fluid; Con = control. CHEST 2015 147, 969-978DOI: (10.1378/chest.14-1426) Copyright © 2015 The American College of Chest Physicians Terms and Conditions

Figure 2 ARDS is associated with increased apelin and APJ receptor expression. Rats were injected with saline or OA. Lungs tissue levels of apelin preproprotein and APJ receptor protein were determined. A, Western blot photographs show ARDS causes increases in apelin preproprotein and APJ receptor protein expression. B and C, Western blot protein bands were quantified using densitometry and were expressed as apelin/β-tubulin or APJ/β-tubulin ratio. Data are presented as mean ± SD of five rats in each group.*P <.05 compared with Con group. See Figure 1 legend for expansion of abbreviations. CHEST 2015 147, 969-978DOI: (10.1378/chest.14-1426) Copyright © 2015 The American College of Chest Physicians Terms and Conditions

Figure 3 ARDS is associated with an increased APJ receptor binding capacity. A, Saturation binding curve for [125I]-apelin-36 binding. Increasing concentrations of [125I]-apelin-36 were incubated with membrane protein (50 μg/tube) from the lungs of the Con and ARDS group rats in the absence (total binding) or presence (nonspecific binding) of 10 μmol/L of unlabeled apelin. Specific binding was determined by subtracting nonspecific binding from total binding. ARDS increased the maximal apelin-binding capacity. B, Scatchard analysis of the apelin binding data shows that ARDS increases the apelin maximal binding capacity without altering the binding affinity. Data are presented as mean ± SD of 10 rats in each group. See Figure 1 legend for expansion of abbreviations. CHEST 2015 147, 969-978DOI: (10.1378/chest.14-1426) Copyright © 2015 The American College of Chest Physicians Terms and Conditions

Figure 4 Apelin augments OA-induced APJ receptor upregulation. Rats were treated with apelin-13 (10 nmol/kg, intraperitoneally [IP]) 1 h before and 2 h after OA injection. Lung tissue levels of APJ protein were measured 6 h after OA injection. A, Western blot photographs show that apelin augments OA-induced APJ receptor protein expression. B, Western blot protein bands were quantified using densitometry and were expressed as APJ/β-tubulin ratio. Data are presented as mean ± SD of five rats in each group.*P <.05 compared with Con group;#P <.05 compared with ARDS group. See Figure 1 legend for expansion of other abbreviations. CHEST 2015 147, 969-978DOI: (10.1378/chest.14-1426) Copyright © 2015 The American College of Chest Physicians Terms and Conditions

Figure 5 Activation of the apelin-APJ signaling protects against OA-induced ARDS. Rats in the Con or ARDS group were injected with saline or OA. Rats in the ARDS + apelin or ARDS + Ala group were injected with apelin-13, APJ receptor agonist, or [Ala]-apelin-13, APJ receptor antagonist (both at 10 nmol/kg, IP), 1 h before and 2 h after OA injection. Lung histology was evaluated, and lung wet/dry weight ratio and BALF protein content were determined 6 h after saline or OA injection. A, Representative photographs of lung sections show that APJ receptor agonist alleviates, and APJ receptor antagonist exacerbates, OA-induced lung pathologies (hematoxylin and eosin, original magnification × 200). Lung section from ARDS rats exhibited increased inflammatory cell infiltration, increased alveolar wall thickness, edema, and patchy areas of hemorrhage, characteristics of OA-induced ARDS. These histologic changes were mitigated by treatment with apelin-13 (ARDS + apelin) and were exacerbated by treatment with [Ala]-apelin-13 (ARDS + Ala). Scale bar, 100 μm. B, APJ receptor agonist alleviates, and APJ receptor antagonist exacerbates, OA-induced lung edema as measured by lung wet/dry ratio. C, APJ receptor agonist alleviates, and APJ receptor antagonist tends to exacerbate, OA-induced increase in lung capillary-alveolar leakage as measured by BALF protein content. Data are presented as mean ± SD of 10 rats in each group.*P <.05 compared with Con group;#P <.05 compared with ARDS group. Ala = [Ala]-apelin-13. See Figure 1 and 4 legends for expansion of other abbreviations. CHEST 2015 147, 969-978DOI: (10.1378/chest.14-1426) Copyright © 2015 The American College of Chest Physicians Terms and Conditions

Figure 6 Activation of the apelin-APJ signaling inhibits OA-induced lung inflammation. Rats in the Con, ARDS, ARDS + apelin, and ARDS + Ala groups were injected as described previously. Lung tissue and BALF levels of TNF-α, MCP-1, and MPO activity were measured. A and D, APJ receptor agonist (ARDS + apelin) inhibits, and APJ receptor antagonist (ARDS + Ala) augments, OA-induced lung tissue (A) and BALF (D) levels of TNF-α. B and E, APJ receptor agonist inhibits, and APJ receptor antagonist augments, OA-induced lung tissue level of MCP-1 (B), and APJ receptor antagonist augments OA-induced BALF level of MCP-1 (E). C and F, APJ receptor agonist inhibits, and APJ receptor antagonist augments, OA-induced lung tissue level of MPO activity (C), and APJ receptor agonist inhibits OA-induced BALF level of MPO activity (F). Data are presented as mean ± SD of 10 rats in each group.*P <.05 compared with Con group;#P <.05 compared with ARDS group. MCP = monocyte chemoattractant protein; MPO = myeloperoxidase; TNF-α = tumor necrosis factor-α. See Figure 1 and 5 legends for expansion of other abbreviations. CHEST 2015 147, 969-978DOI: (10.1378/chest.14-1426) Copyright © 2015 The American College of Chest Physicians Terms and Conditions

Figure 7 Apelin-APJ signaling alleviates oxidant stress and enhances antioxidant mechanism. Rats in the Con, ARDS, ARDS + apelin, and ARDS + Ala groups were injected as described previously. Plasma and lung tissue levels of MDA, a marker of tissue oxidant stress, or lung tissue and BALF levels of GSH, a major antioxidant, were measured. A and B, APJ receptor agonist attenuates, and APJ receptor antagonist augments, OA-induced lung tissue (A) and plasma (B) levels of MDA. C, APJ receptor antagonist attenuates OA-induced lung tissue level of GSH. D, APJ receptor agonist augments, and APJ receptor antagonist attenuates, OA-induced BALF level of GSH. Data are presented as mean ± SD of 10 rats in each group.*P <.05 compared with Con group;#P <.05 compared with ARDS group. GSH = glutathione; MDA = malondialdehyde. See Figure 1 and 5 legends for expansion of other abbreviations. CHEST 2015 147, 969-978DOI: (10.1378/chest.14-1426) Copyright © 2015 The American College of Chest Physicians Terms and Conditions

Figure 8 Postinjury treatment with apelin-13 attenuates OA-induced lung injury. Rats in the Con and ARDS groups were injected with saline or OA (0.2 mL/kg, IV). Rats in the ARDS + apelin group were injected with apelin-13 (10 nmol/kg, IP) 1 h after OA injection, when animals showed clear signs of respiratory distress, and 3 h after the initial dose of apelin-13. Lung histology, lung wet/dry weight ratio, and BALF protein content were assessed 6 h after OA injection. A, Representative photographs of lung sections show that postinjury treatment with apelin-13 ameliorates OA-induced lung pathologies (hematoxylin and eosin, original magnification × 200, scale bar 100 μm). B, Postinjury apelin-13 treatment reduces OA-induced lung edema as measured by lung wet/dry ratio. C, Postinjury apelin-13 treatment decreases OA-induced lung capillary-alveolar leakage as measured by BALF protein content. Data are presented as mean ± SD of seven rats in each group.*P <.05 compared with Con group;#P <.05 compared with ARDS group. See Figure 1 and 4 legends for expansion of abbreviations. CHEST 2015 147, 969-978DOI: (10.1378/chest.14-1426) Copyright © 2015 The American College of Chest Physicians Terms and Conditions

Figure 9 Postinjury treatment with apelin-13 attenuates LPS-induced lung injury. Rats in the Con and LPS groups were intratracheally instilled with saline or LPS (5 mg/kg) and were injected with apelin (10 nmol/kg, IP) 4 h after LPS installation and every 6 h after the initial dose of apelin-13. Lung histology, lung wet/dry weight ratio, and BALF protein content were evaluated 24 h after LPS. A, Representative photographs of lung sections show that postinjury treatment with apelin-13 ameliorates LPS-induced lung pathologies (hematoxylin and eosin, original magnification × 200, scale bar, 100 μm). B, Postinjury apelin-13 treatment reduces LPS-induced lung edema as measured by lung wet/dry ratio. C, Postinjury apelin-13 treatment decreases LPS-induced lung capillary-alveolar leakage as measured by BALF protein content. Data are presented as mean ± SD of six rats in each group.*P <.05 compared with Con group;#P <.05 compared with LPS group. LPS = lipopolysaccharide. See Figure 1 and 4 legends for expansion of other abbreviations. CHEST 2015 147, 969-978DOI: (10.1378/chest.14-1426) Copyright © 2015 The American College of Chest Physicians Terms and Conditions