The Apelin-APJ Axis Is an Endogenous Counterinjury Mechanism in Experimental Acute Lung Injury

[

Original Research Critical Care Medicine

]

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; and Yong-Sheng Gong, MD, PhD

Although the mechanisms and pathways mediating ARDS have been studied extensively, less attention has been given to the mechanisms and pathways that counteract injury responses. This study found that the apelin-APJ pathway is an endogenous counterinjury mechanism that protects against ARDS.

BACKGROUND:

Using a rat model of oleic acid (OA)-induced ARDS, the effects of ARDS on apelin and APJ receptor expressions and on APJ receptor binding capacity were examined. The protective effect of activating the apelin-APJ pathway against OA- or lipopolysaccharide (LPS)induced ARDS was evaluated. METHODS:

RESULTS: ARDS was coupled to upregulations of the apelin and APJ receptor. Rats with OA-induced ARDS had higher lung tissue levels of apelin proprotein and APJ receptor expressions; elevated plasma, BAL fluid (BALF), and lung tissue levels of apelin-36 and apelin-12/13; and an increased apelin-APJ receptor binding capacity. Upregulation of the apelin-APJ system has important pathophysiologic function. Stimulation of the apelin-APJ signaling using receptor agonist apelin-13 alleviated, whereas inhibition of the apelin-APJ signaling using receptor antagonist [Ala]-apelin-13 exacerbated, OA-induced lung pathologies, extravascular lung water accumulation, capillary-alveolar leakage, and hypoxemia. The APJ receptor agonist inhibited, and the APJ receptor antagonist augmented, OA-induced lung tissue and BALF levels of tumor necrosis factor-a and monocyte chemoattractant protein-1, and plasma and lung tissue levels of malondialdehyde. Postinjury treatment with apelin-13 alleviated lung inflammation and injury and improved oxygenation in OA- and LPS-induced lung injury.

The apelin-APJ signaling pathway is an endogenous anti-injury and organprotective mechanism that is activated during ARDS to counteract the injury response and to CHEST 2015; 147(4):969-978 prevent uncontrolled lung injury.

CONCLUSIONS:

Manuscript received June 12, 2014; revision accepted October 13, 2014; originally published Online First November 6, 2014. ABBREVIATIONS: BALF 5 BAL fluid; GSH 5 glutathione; IP 5 intraperitoneally; LPS 5 lipopolysaccharide; MDA 5 malondialdehyde; MPO 5 myeloperoxidase; OA 5 oleic acid; PAH 5 pulmonary arterial hypertension; Sao2 5 arterial blood oxygen saturation AFFILIATIONS: From the Institute of Hypoxia Medicine (Drs Fan, Xue, Zhang, Xing, H. Liu, Mao, Kong, S. F. Liu, and Gong), Wenzhou Medical University, Zhejiang, China; the Department of Pathophysiology (Dr Gao), Third Military Medical University, Chongqing, China; and The Feinstein Institute for Medical Research (Dr S. F. Liu), Manhasset, NY. FUNDING/SUPPORT: This work was supported by the National Nature Science Foundation of China [81370171, 81200039], the Natural Science

Foundation of Zhejiang Province of China [Y2091033], the Major State Basic Research Development Program of China [2012CB518200], and the Science and Technology Foundation of Wenzhou, China [Y20060059]. CORRESPONDENCE TO: Yong-Sheng Gong, MD, PhD, Institute of Hypoxia Medicine, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China; e-mail: [email protected] © 2015 AMERICAN COLLEGE OF CHEST PHYSICIANS. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.14-1426

journal.publications.chestnet.org

Downloaded From: http://journal.publications.chestnet.org/ by a University of Pittsburgh User on 06/03/2015

969

Acute lung injury and ARDS are devastating consequences of many inflammatory and traumatic conditions, including pneumonia, sepsis, trauma, aspiration, pancreatitis, and severe burn.1,2 Injurious insults trigger cascades of molecular and cellular events, leading to the activation of inflammatory and injury pathways, the activation of leukocytes and platelets, the generation of reactive oxidant species, the release of proteases, and the activation of coagulation pathways.3-5 These mechanisms act in concert to cause inflammation, increased lung endothelial and epithelial permeability, pulmonary extravascular accumulation of protein-rich fluid, and hypoxia, which are cardinal characteristics of ARDS.1-5 In addition to inflammatory and injury pathways, the lungs are endowed with tissue-protective mechanisms.6,7 These mechanisms counteract the actions of inflammatory and injurious mediators and prevent uncontrolled lung inflammation and injury. Activation of inflammatory and injury pathways may be the driving force for developing ARDS. However, it is the delicate balance between proand antiinflammatory factors, and between injury and counterinjury mechanisms, that may determine the extent of lung inflammation and injury and clinical outcomes. Thus, uncovering new mechanisms that protect against ARDS is significant in that it will not only improve our understanding of the pathogenic mechanisms of ARDS, but will also identify a new target for developing therapy to combat ARDS. Apelin is a group of small peptides derived from a common precursor, preproapelin. Several active apelin peptides, including apelin-36, apelin-13 and apelin-12, have been reported.8,9 All apelin peptides exert their biologic effects by binding to a G-proteincoupled receptor, the APJ receptor, leading to biologic responses.8,9 Apelin and its receptor have a wide range

Materials and Methods Animal Experiments All animal studies were approved by the animal care and use committee of Wenzhou Medical University and complied with US National Institutes of Health guidelines. Male rats (of about 250 g) were randomly divided into six groups (five to 10 per group). Rats in the control (Con) or ARDS group were injected with saline (0.2 mL/kg, IV) or OA (0.2 mL/kg, IV). Rats in the Con 1 apelin or Con 1 Ala group, or in the ARDS 1 apelin or ARDS 1 Ala group, were injected with apelin-13 or [Ala]-apelin-13 (both at 10 nmol/kg, intraperitoneally [IP])

970 Original Research

of tissue distribution and have diverse physiologic functions.8,9 Apelin causes vasodilatation,10 lowers BP,11 antagonizes angiotensin- or vasopressin-induced vasoconstriction,9,12 and improves cardiac contractility.13 Apelin-APJ signaling regulates the embryonic development of the cardiovascular system14 and modulates angiogenesis and neovascularization in adult mice.15,16 The apelin and APJ receptor are upregulated during tissue injury.17-21 However, the role of this upregulation varies with the organs. Apelin upregulation protects against tissue injury in the heart and pancreas17-19 but mediates tissue injury in the liver and dorsal root.20,21 Levels of apelin and APJ receptor expressions are high in the lungs22 but are downregulated under some pathologic conditions. Patients and animals with pulmonary arterial hypertension (PAH) have lower plasma apelin levels and a reduced apelin expression in lung endothelial cells23 or lung tissue,24 which may contribute to the development of PAH.23-25 In a rat model of 100% oxygen exposureinduced bronchopulmonary dysplasia, apelin is up- or downregulated depending on cell type.26 Despite all this evidence, the effects of ARDS on apelin and APJ receptor expression, and the modulatory role of the apelin-AJP system in ARDS, have not been studied. In this study, we tested the hypothesis that the apelinAPJ system serves as an endogenous counterinjury mechanism in a rat model of oleic acid (OA)-induced ARDS. We chose an OA-induced ARDS model for two reasons: (1) OA-induced ARDS exhibits histopathologic and physiologic features that are similar to those of human ARDS27 and (2) OA-induced ARDS is severe, which allows us to reliably test the protective effects of the apelin-APJ system. We demonstrated, we believe for the first time, that apelin-APJ signaling is activated during ARDS, and that it serves as an endogenous counterinjury mechanism that protects against experimental ARDS.

(Phoenix Pharmaceuticals, Inc) 1 h before and 2 h after saline or OA injection. In the postinjury treatment studies, apelin (10 nmol/kg, IP) was administered 1 h after OA injection and 3 h after the initial dose of apelin-13. At 6 h after OA injection, arterial blood gas was analyzed. This was followed by BAL, and blood and lung samples were collected. For the lipopolysaccharide (LPS) model, rats were intratracheally instilled with Escherichia coli LPS (5 mg/kg), and were injected with apelin (10 nmol/kg, IP) 4 h after LPS installation, followed by an additional dose of apelin-13 every 6 h. Measurements were made 24 h after LPS. Details of the materials and methods are given in e-Appendix 1.

[

Downloaded From: http://journal.publications.chestnet.org/ by a University of Pittsburgh User on 06/03/2015

147#4 CHEST APRIL 2015

]

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 5 BAL fluid; Con 5 control.

Results OA-Induced ARDS Increases Apelin Protein Level

An increase in the apelin or APJ receptor or both after ARDS may support an anti-injury role. Therefore, we measured plasma, lung, and BAL fluid (BALF) levels of apelin-36 and apelin-12/13, three active apelin fragments. The peptide sequences of apelin-12 and apelin-13 differ by only one amino acid. It is likely that the apelin-12 radioimmunoassay kit detects both peptides. We used apelin-12/13 to describe the apelin-12 measurement result. Rats with OA-induced ARDS had significantly elevated plasma, lung tissue, and BALF levels of apelin-36 and apelin-12/13 (Fig 1). The elevated apelin level appears to be a result of an increased apelin expression. Western blot showed an increased lung tissue level of apelin proprotein in ARDS rats (Fig 2).

Saturation-binding assay and Scatchard plot analysis showed an increased 125I-apelin-36 binding capacity in the membrane-enriched fraction from injured lungs. The maximal number of receptor binding site values were 19.34 ⫾ 1.88 and 30.75 ⫾ 3.11 fmol/mg protein (P , .05), and the dissociation constant values were 50.25 ⫾ 4.12 and 47.25 ⫾ 5.22 nmol/L for the control and ARDS groups, respectively (Fig 3). This represents a 59% increase in

OA-Induced ARDS Increases APJ Receptor Expression and Receptor Binding Capacity

Apelin exerts its effect by binding to the APJ receptor. We examined the effect of OA-induced ARDS on APJ receptor protein level and activity. We prepared membrane protein from lungs and confirmed the purity of the membrane-enriched fraction by demonstrating that Na1/K1-ATPase activity, a plasma membrane marker, was 5.7-fold higher in the membrane fraction than in the total lung homogenates (6.3 ⫾ 0.7 mol/mg protein vs 0.94 ⫾ 0.2 mol/mg protein, P , .01). Western blot demonstrated an increased APJ receptor protein level in injured lungs, compared with control lungs (Fig 2).

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/b-tubulin or APJ/b-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.

journal.publications.chestnet.org

Downloaded From: http://journal.publications.chestnet.org/ by a University of Pittsburgh User on 06/03/2015

971

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 mg/tube) from the lungs of the Con and ARDS group rats in the absence (total binding) or presence (nonspecific binding) of 10 mmol/L of unlabeled apelin. Specific binding was determined by subtracting nonspecific binding from total binding. ARDS increased the maximal apelinbinding 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.

the maximal number of receptor binding sites in injured lungs, although the dissociation constant values were similar between the two groups. This result illustrates that OA-induced ARDS upregulates apelin and its receptor in the lungs. There appears to be a positive autoregulation, because apelin-13 augmented ARDSinduced APJ receptor protein expression (Fig 4). Blockade of Apelin-APJ Pathway Exacerbates OA-Induced ARDS

To clarify the physiologic and pathophysiologic significance of apelin-APJ upregulation, we examined the

effects of blocking the apelin-APJ signaling on OA-induced ARDS. We inhibited apelin-APJ signaling using [Ala]apelin-13, which is a competitive APJ receptor antagonist.11 Rats injected with OA developed obvious ARDS, as indicated by histopathologic changes in the lungs, including increased inflammatory cell infiltration, increased alveolar wall thickness, edema, and patchy areas of hemorrhage (Fig 5A). The histologic changes were associated with pathophysiologic changes, including hypoxemia (Table 1), extravascular lung water accumulation (Fig 5B), protein-rich fluid leakage into airspaces (Fig 5C), and increased BALF cell counts (Table 2). Rats injected with OA plus [Ala]-apelin-13, given 1 h before and 2 h after OA injection, exhibited more severe histopathologic changes in the lungs (Fig 5A), a higher lung wet/dry weight ratio (Fig 5B) and lower Pao2 (Table 1), compared with rats injected with OA alone. Administration of [Ala]-apelin-13 to control rats had no effect (e-Fig 1). This result suggests that inhibition of the apelin-APJ signaling exacerbates OA-induced ARDS. Stimulation of Apelin-APJ Pathway Alleviates OA-Induced ARDS

To enforce the notion that the apelin-APJ signaling is an endogenous anti-injury mechanism, we examined whether the APJ receptor agonist apelin-13 alleviates OA-induced ARDS. Histologic examination showed that treatment of OA-injected rats with apelin-13, given 1 h before and 2 h after OA injection, ameliorated virtually all major histopathologic changes induced by OA in the lungs (Fig 5A). Treatment of OA-injected rats with apelin-13 decreased the lung wet/dry weight ratio (Fig 5B), reduced BALF cell counts (Table 2), reduced fluid leakage into airspaces (Fig 5C), increased Pao2, and tended to increase arterial blood oxygen saturation (Sao2)% (Table 1). Treatment of control rats with apelin-13 had no effect (e-Fig 1). This result illustrates that activation of the apelin-APJ pathway alleviates OA-induced ARDS. Aapelin-13 Attenuates and [Ala]-Apelin-13 Potentiates OA-Induced Lung Inflammation

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/b-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.

972 Original Research

Inflammation is a major mechanism of all types of lung injury.1-4,27 We examined the effect of stimulating or inhibiting the apelin-APJ signaling by administering apelin-13 or [Ala]-apelin-13 1 h before and 2 h after OA injection, on OA-induced inflammatory cytokine production. Compared with control rats, rats injected with OA had significantly increased lung tissue and BALF levels of tumor necrosis factor-a and monocyte chemoattractant protein-1 (Fig 6). Apelin-13 significantly

[

Downloaded From: http://journal.publications.chestnet.org/ by a University of Pittsburgh User on 06/03/2015

147#4 CHEST APRIL 2015

]

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 1 apelin or ARDS 1 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 3 200). 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 1 apelin) and were exacerbated by treatment with [Ala]-apelin-13 (ARDS 1 Ala). Scale bar, 100 mm. 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 5 [Ala]-apelin-13. See Figure 1 and 4 legends for expansion of other abbreviations.

attenuated, and [Ala]-apelin-13 significantly potentiated, OA-induced increases in cytokine levels (Fig 6). Rats injected with OA had higher lung tissue and BALF levels of myeloperoxidase (MPO) activity, a marker of neutrophil infiltration, which was attenuated by treatment with apelin-13 and potentiated by treatment with [Ala]-apelin (Figs 6C, 6F). Administration of apelin-13 or [Ala]apelin-13 to control rats had no effect on cytokines or MPO activity (e-Fig 2). Thus, activation of the apelinAPJ system attenuates OA-induced lung inflammation. TABLE 1

Aapelin-13 Attenuates and [Ala]-Apelin-13 Potentiates OA-Induced Oxidant Stress

Oxidant stress plays an important role in the pathogenesis of ARDS.5 Glutathione (GSH) is a major endogenous antioxidant that directly neutralizes free radicals. We evaluated the effect on tissue oxidant stress of apelin-13 or [Ala]-apelin-13, given 1 h before and 2 h after OA injection. As expected, rats injected with OA had remarkably higher plasma and lung tissue levels of malondialdehyde (MDA), a marker of oxidant stress

] Activation of Apelin-APJ Signaling Improves Oxygenation

Group

No.

pH

PaO2, mm Hg

PaCO2, mm Hg

SaO2, %

Control

10

7.39 ⫾ 0.03

97.6 ⫾ 3.2

43.0 ⫾ 6.3

96.5 ⫾ 1.3

ARDS

10

7.41 ⫾ 0.04

61.9 ⫾ 7.7a

43.2 ⫾ 3.2

88.2 ⫾ 2.1

ARDS 1 apelin

10

7.42 ⫾ 0.04

78.4 ⫾ 5.8b

43.7 ⫾ 3.0

93.7 ⫾ 2.0

ARDS 1 Ala

10

7.37 ⫾ 0.02

50.5 ⫾ 8.5a,b

46.2 ⫾ 2.8

83.3 ⫾ 7.5a

Data are presented as mean ⫾ SD of 10 rats per group. Apelin-13, APJ receptor agonist, or Ala, APJ receptor antagonist, was injected 1 h before and 2 h after oleic acid (OA) injection. Arterial blood gas was analyzed 6 h after OA injection. Ala 5 [Ala]-apelin-13; SaO2 5 arterial blood oxygen saturation. aP , .05 compared with control group. bP , .05 compared with ARDS group.

journal.publications.chestnet.org

Downloaded From: http://journal.publications.chestnet.org/ by a University of Pittsburgh User on 06/03/2015

973

TABLE 2

] Activation of Apelin-APJ Signaling Reduces BALF Cell Counts

Group

No.

Total Cell Counts, 3 104

Neutrophils, 3 104

Control

10

7.88 ⫾ 1.96

0.87 ⫾ 0.12

ARDS

10

121.60 ⫾ 16.69a

ARDS 1 apelin

10

64.75 ⫾ 13.83

ARDS 1 Ala

10

125.21 ⫾ 10.54a

b

88.65 ⫾ 12.35a 32.78 ⫾ 5.38b 93.74 ⫾ 14.22a

Data are presented as mean ⫾ SD of 10 rats per group. Apelin-13 or Ala was injected 1 h before and 2 h after OA injection. BAL fluid total cells and neutrophils were counted 6 h after OA injection. See Table 1 legend for expansion of abbreviations. aP , .05 compared with control group. bP , .05 compared with ARDS group.

(Figs 7A, 7B). Apelin-13 significantly inhibited, and [Ala]apelin-13 augmented, OA-induced elevation in plasma and lung tissue MDA levels (Figs 7A, 7B). To counterbalance the oxidant stress, lung and BALF levels of GSH were also elevated in OA-injected rats. Apelin-13 potentiated the OA-induced elevation in GSH level in BALF, whereas [Ala]-apelin-13 attenuated the OA-induced elevation in GSH level in the lungs and BALF (Figs 7C, 7D). Apelin-13 or [Ala]-apelin-13 had no effect on

MDA or GSH in control rats (e-Fig 3). Thus, activation of the apelin-APJ pathway alleviates OA-induced oxidant stress and enhances the antioxidant mechanism. Postinjury Treatment With Apelin-13 Alleviates Lung Inflammation and Injury

To explore therapeutic potential, OA- or LPS-challenged rats were administered apelin-13 1 h after OA injection, when animals showed clear signs of respiratory distress, or 4 h after LPS instillation. Lung inflammation and injury were assessed at 6 or 24 h after OA or LPS injection. Apelin-13 treatment attenuated all major histologic changes, lowered the wet/dry weight ratio, reduced alveolar capillary leakage (Figs 8, 9), attenuated inflammation (e-Figs 4, 5) in the lungs, and improved oxygenation (Tables 3, 4) in both OA- and LPS-induced ARDS models. This result suggests a therapeutic potential of activating the apelin-APJ pathway for treating ARDS.

Discussion Although the mechanisms and pathways that mediate lung inflammation and injury have been studied extensively, the mechanisms and pathways that protect

Figure 6 – Activation of the apelin-APJ signaling inhibits OA-induced lung inflammation. Rats in the Con, ARDS, ARDS 1 apelin, and ARDS 1 Ala groups were injected as described previously. Lung tissue and BALF levels of TNF-a, MCP-1, and MPO activity were measured. A and D, APJ receptor agonist (ARDS 1 apelin) inhibits, and APJ receptor antagonist (ARDS 1 Ala) augments, OA-induced lung tissue (A) and BALF (D) levels of TNF-a. 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 5 monocyte chemoattractant protein; MPO 5 myeloperoxidase; TNF-a 5 tumor necrosis factor-a. See Figure 1 and 5 legends for expansion of other abbreviations.

974 Original Research

[

Downloaded From: http://journal.publications.chestnet.org/ by a University of Pittsburgh User on 06/03/2015

147#4 CHEST APRIL 2015

]

and improved oxygenation in both OA- and LPS-induced ARDS models. Taken together, we have demonstrated that ARDS is coupled to activation of the apelin-APJ pathway, which counteracts inflammatory and injury responses, preventing uncontrolled lung injury.

Figure 7 – Apelin-APJ signaling alleviates oxidant stress and enhances antioxidant mechanism. Rats in the Con, ARDS, ARDS 1 apelin, and ARDS 1 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 5 glutathione; MDA 5 malondialdehyde. See Figure 1 and 5 legends for expansion of other abbreviations.

against lung inflammation and injury have been less well studied. As a consequence, few endogenous anti-injury mechanisms in the lungs have been reported.6,7 Here, we demonstrate that the apelin-APJ signaling pathway is an endogenous counterinjury mechanism that protects against lung injury. We found that OA-induced ARDS was associated with an increased apelin proprotein expression in the lungs and elevated plasma, BALF, and lung tissue levels of apelin-36 and apelin-12/13. OA-induced ARDS increased APJ receptor protein expression and receptor binding capacity. There appears to be a positive autoregulation, because apelin-13, the ligand for the AJP receptor, augmented ARDS-induced upregulation of APJ receptor expression. The enhanced apelin-APJ signaling has important pathophysiologic function. Stimulation of the apelin-APJ signaling using receptor agonist apelin-13 alleviated, and repression of the apelin-APJ signaling using receptor antagonist [Ala]-apelin-13, exacerbated OA-induced lung pathologies, lung extravascular water accumulation, capillaryalveolar leakage, and hypoxemia. Postinjury treatment with apelin-13 attenuated lung inflammation and injury

Antiinflammation may be one mechanism by which apelin-APJ signaling exerts its organ-protective effect. In parallel with alleviation or exacerbation of OA-induced lung injury, the APJ receptor agonist attenuated, or the APJ receptor antagonist potentiated, lung inflammation. Apelin-13 decreased lung tissue and BALF levels of inflammatory cytokines and reduced the lung tissue level of MPO activity in OA-injected rats. [Ala]-apelin-13 had the opposite effect. Inflammation is a well-established mechanism of ARDS.1-4 Others have shown that apelin inhibited nuclear factor-kB activity.19 Thus, inhibition of lung inflammation at least partially explains the organ-protective action of the apelin-APJ signaling. Consistent with the reduction in MPO activity, apelin-13 reduced BALF total and neutrophil counts. The antiinflammatory action of apelin may partially explain the inhibition by apelin-13 of inflammatory cell accumulation in the airspaces. The anti-injury action of apelin may also contribute to the reduction in BALF cell count. OA infusion causes endothelial and epithelial damage, detachment, and necrosis,27 all of which allow blood cells to gain easy access to airspaces. Apelin alleviates endothelial and epithelial damage, reduces the access, and subsequently decreases blood cell accumulation in airspaces. Neutrophil traffic in the lungs is influenced by hemodynamic factors. The vasodilator effect of apelin may contribute to the decreased neutrophil accumulation in the airspaces. Regulation of oxidant and antioxidant balance may be another mechanism by which the apelin-APJ signaling protects against lung injury. Oxidant stress is a common mechanism of lung injury.5 Reactive oxidant species directly modify proteins, lipids, carbohydrates, and DNA, causing damage and aberrant functions of those molecules.5 Oxidative stress activates redox-sensitive transcription factors, such as nuclear factor-kB, which mediate the expression of inflammatory cytokines that further aggravates lung inflammation and injury.28 We showed that apelin-13 concomitantly alleviated OA-induced lung tissue oxidant stress and injury, whereas [Ala]-apelin-13 augmented OA-induced lung tissue oxidant stress associated with an exacerbated lung injury. We further demonstrated that apelin-13 enhanced,

journal.publications.chestnet.org

Downloaded From: http://journal.publications.chestnet.org/ by a University of Pittsburgh User on 06/03/2015

975

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 1 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 3 200, scale bar 100 mm). 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 capillaryalveolar 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.

and [Ala]-apelin-13 attenuated, OA-induced elevation in the BALF level of GSH, which represents a compensatory antioxidant activity that counterbalances oxidant stress. Others have reported that apelin suppressed oxidant stress and stimulated catalase activity in the heart.29

Taken together, our data suggest that activation of the apelin-APJ signaling represses OA-induced oxidant stress and enhances the antioxidant mechanism in the lungs, which may contribute to the organ-protective action of the apelin-APJ signaling. 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 3 200, scale bar, 100 mm). 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 5 lipopolysaccharide. See Figure 1 and 4 legends for expansion of other abbreviations.

976 Original Research

[

Downloaded From: http://journal.publications.chestnet.org/ by a University of Pittsburgh User on 06/03/2015

147#4 CHEST APRIL 2015

]

TABLE 3

] Postinjury Treatment With Apelin-13 Improves Oxygenation in OA Model

Group

No.

pH

PaO2, mm Hg

PaCO2, mm Hg

SaO2, %

Control

7

7.412 ⫾ 0.009

85.7 ⫾ 7.94

44.68 ⫾ 3.41

96.83 ⫾ 1.94

ARDS

7

7.447 ⫾ 0.042

47.6 ⫾ 10.01a

44.46 ⫾ 6.70

82.20 ⫾ 12.03a

ARDS 1 apelin

7

7.456 ⫾ 0.029

68.0 ⫾ 13.10b

39.32 ⫾ 4.42

93.20 ⫾ 3.701b

Data are presented as mean ⫾ SD. Apelin-13 was injected 1 h after OA injection, when animals showed clear signs of respiratory distress, and 3 h after the initial dose of apelin-13. Arterial blood gas was analyzed 6 h after OA injection. See Table 1 legend for expansion of abbreviations. aP , .05 compared with control group. bP , .05 compared with ARDS group.

The molecular mechanisms mediating the antiinflammatory, antioxidant, and organ-protective effects of the apelin-APJ axis remain to be elucidated. One potential pathway downstream of the APJ receptor is the nitric oxide pathway. In a rat model of bronchopulmonary dysplasia, prophylactic treatment of rats with apelin reduced inflammation, pulmonary remodeling, and right ventricular hypertrophy.26 Cotreatment of rats with nitric oxide synthase inhibitor abolished all the beneficial effects of apelin, highlighting the important role of the nitric oxide pathway in mediating the effects of apelin.26 Other downstream signaling pathways may also be important and warrant further investigation.8,9

showing that apelin protects against organ injury in the heart and pancreas,17-19 but is in contrast to the finding of other reports demonstrating that apelin mediates tissue injury in the liver and dorsal root.20,21 The apelinAPJ signaling appears to play organ-dependent roles in tissue injury.

The current study builds on earlier studies. Prior studies have shown that apelin and/or the APJ receptor are upregulated during tissue injury.17-21 However, the effect of this upregulation was reported to be beneficial in some organs17-19 but detrimental in others.20,21 The role this pathway plays in ARDS remains unknown. Apelin expression is downregulated in patients and animals with PAH, which may contribute to the development of PAH.2325 These prior findings prompted us to examine the role of the apelin-APJ signaling in ARDS. Our study builds on the findings of previous reports by presenting the first evidence that the apelin-APJ signaling serves as an endogenous anti-injury mechanism against lung injury. This finding is consistent with that of previous reports

Conclusions

TABLE 4

This study has limitations. Although we demonstrated that the apelin-APJ pathway is an organ-protective mechanism, the molecular mechanisms underlying apelin’s protective action remain to be elucidated. The short plasma half-life of apelin may present a limitation in translating the current finding into clinical therapy. Development of a long-acting APJ receptor agonist will overcome this limitation. In summary, OA-induced ARDS was coupled to upregulation and activation of the apelin-APJ pathway. The enhanced apelin-APJ signaling plays a functional role. Stimulation of the apelin-APJ signaling alleviated, and repression of the apelin-APJ signaling exacerbated, OA-induced lung inflammation and injury. Postinjury treatment with apelin-13 attenuated lung injury and improved oxygenation in both OA- and LPS-induced ARDS models. Taken together, we have demonstrated that ARDS is coupled to an activation of the apelinAPJ signaling pathway, which counteracts inflammatory and injury responses, and prevents uncontrolled ARDS.

] Postinjury Treatment With Apelin-13 Improves Oxygenation in LPS Model No.

pH

PaO2, mm Hg

PaCO2, mm Hg

SaO2, %

Control

Group

6

7.414 ⫾ 0.008

88.80 ⫾ 2.28

43.82 ⫾ 2.99

97.40 ⫾ 1.52

LPS

6

7.368 ⫾ 0.027a

63.67 ⫾ 10.35a

57.87 ⫾ 6.22a

LPS 1 apelin

6

7.423 ⫾ 0.013b

77.50 ⫾ 12.34b

49.63 ⫾ 3.86b

89.5 ⫾ 7.18a 94.67 ⫾ 2.73

Data are presented as mean ⫾ SD. Apelin-13 was injected 4 h after intratracheal LPS installation, and every 6 h after the initial dose of apelin-13. Arterial blood gas was analyzed 24 h after LPS installation. LPS 5 lipopolysaccharide. See Table 1 legend for expansion of other abbreviations. aP , .05 compared with control group. bP , .05 compared with LPS group.

journal.publications.chestnet.org

Downloaded From: http://journal.publications.chestnet.org/ by a University of Pittsburgh User on 06/03/2015

977

Acknowledgments Author contributions: X.-F. F. and Y.-S. G. had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Y.-S. G. contributed to the study conception and design, supervision of the studies, and the obtaining of funding; X.-F. F., F. X., Y.-Q. Z., X.-P. X., H. L., S.-Z. M., X.-X. K., and Y.-Q. G. contributed to the study design and execution, and the data acquisition, analysis, and interpretation; and S. F. L. contributed to the study conception and drafting of the manuscript. Financial/nonfinancial disclosures: The authors have reported to CHEST that no conflicts of interest exist with any companies/ organizations whose products or services may be discussed in this article. Role of sponsors: The sponsor had no role in the design of the study, the collection and analysis of the data, or the preparation of the manuscript. Additional information: The e-Appendix and e-Figures can be found in the Supplemental Materials section of the online article.

References 1. Matthay MA, Ware LB, Zimmerman GA. The acute respiratory distress syndrome. J Clin Invest. 2012;122(8):2731-2740. 2. Tsushima K, King LS, Aggarwal NR, De Gorordo A, D’Alessio FR, Kubo K. Acute lung injury review. Intern Med. 2009;48(9):621-630. 3. Herold S, Gabrielli NM, Vadász I. Novel concepts of acute lung injury and alveolarcapillary barrier dysfunction. Am J Physiol Lung Cell Mol Physiol. 2013; 305(10):L665-L681. 4. Bhattacharya J, Matthay MA. Regulation and repair of the alveolar-capillary barrier in acute lung injury. Annu Rev Physiol. 2013;75:593-615. 5. Sarma JV, Ward PA. Oxidants and redox signaling in acute lung injury. Compr Physiol. 2011;1(3):1365-1381. 6. Mulligan MS, Warner RL, Foreback JL, Shanley TP, Ward PA. Protective effects of IL-4, IL-10, IL-12, and IL-13 in IgG immune complex-induced lung injury: role of endogenous IL-12. J Immunol. 1997;159(7):3483-3489.

978 Original Research

7. Yan C, Ward PA, Wang X, Gao H. Myeloid depletion of SOCS3 enhances LPS-induced acute lung injury through CCAAT/enhancer binding protein d pathway. FASEB J. 2013;27(8):2967-2976. 8. Kleinz MJ, Davenport AP. Emerging roles of apelin in biology and medicine. Pharmacol Ther. 2005;107(2):198-211. 9. Galanth C, Hus-Citharel A, Li B, LlorensCortès C. Apelin in the control of body fluid homeostasis and cardiovascular functions. Curr Pharm Des. 2012;18(6): 789-798. 10. Zhong JC, Yu XY, Huang Y, Yung LM, Lau CW, Lin SG. Apelin modulates aortic vascular tone via endothelial nitric oxide synthase phosphorylation pathway in diabetic mice. Cardiovasc Res. 2007;74(3): 388-395. 11. Lee DK, Saldivia VR, Nguyen T, Cheng R, George SR, O’Dowd BF. Modification of the terminal residue of apelin-13 antagonizes its hypotensive action. Endocrinology. 2005;146(1):231-236. 12. Chun HJ, Ali ZA, Kojima Y, et al. Apelin signaling antagonizes Ang II effects in mouse models of atherosclerosis. J Clin Invest. 2008;118(10):3343-3354. 13. Ashley EA, Powers J, Chen M, et al. The endogenous peptide apelin potently improves cardiac contractility and reduces cardiac loading in vivo. Cardiovasc Res. 2005;65(1):73-82. 14. Kang Y, Kim J, Anderson JP, et al. Apelin-APJ signaling is a critical regulator of endothelial MEF2 activation in cardiovascular development. Circ Res. 2013;113(1):22-31. 15. Eyries M, Siegfried G, Ciumas M, et al. Hypoxia-induced apelin expression regulates endothelial cell proliferation and regenerative angiogenesis. Circ Res. 2008; 103(4):432-440. 16. Kasai A, Ishimaru Y, Kinjo T, et al. Apelin is a crucial factor for hypoxia-induced retinal angiogenesis. Arterioscler Thromb Vasc Biol. 2010;30(11):2182-2187. 17. Rastaldo R, Cappello S, Folino A, Losano G. Effect of apelin-apelin receptor system in postischaemic myocardial protection: a pharmacological postconditioning tool? Antioxid Redox Signal. 2011;14(5):909-922. 18. Zeng XJ, Zhang LK, Wang HX, Lu LQ, Ma LQ, Tang CS. Apelin protects heart

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

[

Downloaded From: http://journal.publications.chestnet.org/ by a University of Pittsburgh User on 06/03/2015

against ischemia/reperfusion injury in rat. Peptides. 2009;30(6):1144-1152. Han S, Englander EW, Gomez GA, et al. Pancreatitis activates pancreatic apelinAPJ axis in mice. Am J Physiol Gastrointest Liver Physiol. 2013;305(2):G139-G150. Yasuzaki H, Yoshida S, Hashimoto T, et al. Involvement of the apelin receptor APJ in Fas-induced liver injury. Liver Int. 2013;33(1):118-126. Lang L, Ingorokva S, Hausott B, et al. Selective up-regulation of the vasodilator peptide apelin after dorsal root but not after spinal nerve injury. Neuroscience. 2010;170(3):954-960. Hosoya M, Kawamata Y, Fukusumi S, et al. Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin. J Biol Chem. 2000;275(28): 21061-21067. Andersen CU, Hilberg O, Mellemkjær S, Nielsen-Kudsk JE, Simonsen U. Apelin and pulmonary hypertension. Pulm Circ. 2011;1(3):334-346. Falcão-Pires I, Gonçalves N, HenriquesCoelho T, Moreira-Gonçalves D, Roncon-Albuquerque R Jr, Leite-Moreira AF. Apelin decreases myocardial injury and improves right ventricular function in monocrotaline-induced pulmonary hypertension. Am J Physiol Heart Circ Physiol. 2009;296(6):H2007-H2014. Chandra SM, Razavi H, Kim J, et al. Disruption of the apelin-APJ system worsens hypoxia-induced pulmonary hypertension. Arterioscler Thromb Vasc Biol. 2011;31(4):814-820. Visser YP, Walther FJ, Laghmani H, Laarse Av, Wagenaar GT. Apelin attenuates hyperoxic lung and heart injury in neonatal rats. Am J Respir Crit Care Med. 2010;182(10):1239-1250. Matute-Bello G, Frevert CW, Martin TR. Animal models of acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2008;295(3):L379-L399. Liu SF, Malik ABNF. NF-k B activation as a pathological mechanism of septic shock and inflammation. Am J Physiol Lung Cell Mol Physiol. 2006;290(4):L622-L645. Foussal C, Lairez O, Calise D, et al. Activation of catalase by apelin prevents oxidative stress-linked cardiac hypertrophy. FEBS Lett. 2010;584(11):2363-2370.

147#4 CHEST APRIL 2015

]