j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 6 3 1 e6 3 7
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
C-type natriuretic peptide attenuates lipopolysaccharide-induced acute lung injury in mice Toru Kimura, MD,a,b,* Takashi Nojiri, MD, PhD,a,b Hiroshi Hosoda, MD, PhD,c Shin Ishikane, PhD,a Yasushi Shintani, MD, PhD,b Masayoshi Inoue, MD, PhD,b Mikiya Miyazato, MD, PhD,a Meinoshin Okumura, MD, PhD,b and Kenji Kangawa, PhDa a
Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, Suita-City, Osaka, Japan b Department of General Thoracic Surgery, Osaka University Graduate School of Medicine, Suita-City, Osaka, Japan c Department of Regenerative Medicine and Tissue Engineering, National Cerebral and Cardiovascular Center Research Institute, Suita-City, Osaka, Japan
article info
abstract
Article history:
Background: C-type natriuretic peptide (CNP), secreted by vascular endothelial cells, belongs
Received 30 July 2014
to a family of peptides that includes atrial and brain natriuretic peptides. CNP exhibits
Received in revised form
many vasoprotective effects against pulmonary hypertension and pulmonary fibrosis. The
15 October 2014
objective of this study was to investigate the prophylactic effects of CNP in a mouse model
Accepted 13 November 2014
of lipopolysaccharide (LPS)-induced acute lung injury (ALI).
Available online 20 November 2014
Materials and methods: C57BL/6 mice were divided into three groups as follows: normal control
Keywords:
Twenty-four hours after tail vein injection of LPS, histopathologic, gene expression, and bron-
C-type natriuretic peptide
choalveolar lavage fluid (BALF) assessments were performed on the lungs. To examine the
Acute lung injury
neutrophils in the lungs, cells positive for myeloperoxidase staining were detected by immu-
Pulmonary inflammation
nohistochemistry. BALF cytokine levels were analyzed by enzyme-linked immunosorbent
Anti-inflammation
assays. Gene expression in lung tissue was analyzed by real-time polymerase chain reaction.
mice (n ¼ 13), LPS mice treated with vehicle (n ¼ 12), and LPS mice treated with CNP (n ¼ 12).
Results: CNP significantly attenuated the elevation of leukocyte cell counts and levels of tumor necrosis factor-alpha, macrophage inflammatory protein-2, monocyte chemoattractant protein1, interleukin-6, and keratinocyte-derived chemokine in the BALF after LPS injection. Furthermore, there were significantly fewer myeloperoxidase-positive cells in lungs treated with CNP after LPS injection. In lungs of CNP-treated mice, expression of the monocyte chemoattractant protein-1, S100A8, and E-selectin genes was significantly lower than that in vehicle-treated mice. Conclusions: CNP had a protective effect on ALI induced by LPS by reducing inflammatory cell infiltration. CNP may hold promise in therapeutic strategies for ALI after pulmonary resection surgery. ª 2015 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita-city, Osaka 565 8565, Japan. Tel.: þ81 6 6833 5012; fax: þ81 6 6833 9865. E-mail address: [email protected] (T. Kimura). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.11.023
632
1.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 6 3 1 e6 3 7
Introduction
Acute lung injury (ALI) is a syndrome consisting of acute respiratory failure with bilateral pulmonary infiltrates associated with both pulmonary and nonpulmonary risk factors [1]. ALI is also caused by pulmonary resection surgery because of mechanical and inflammatory stress [2]. Perioperative management of ALI is therefore an important issue for pulmonary resection surgery. Because no prophylactic strategy has been established, the development of effective treatments is desirable. Although there are numerous causes of ALI, endotoxin is thought to be the most common pathogen leading to the development of ALI. Lipopolysaccharide (LPS) is derived from the cell wall of gram-negative bacteria and is known as a major factor contributing to the development of ALI, including the recruitment of inflammatory cells into the lung with subsequent increases in capillary permeability and alveolar edema [3]. C-type natriuretic peptide (CNP), a member of the natriuretic peptide (NP) family, which includes atrial and brain natriuretic peptides (ANP and BNP, respectively), was initially isolated from porcine brain as the third member of the NP family [4]. CNP is secreted by vascular endothelial cells in a wide variety of tissues and has been shown to exhibit a wide range of vasoprotective effects against pulmonary hypertension and pulmonary fibrosis, including antifibrotic, antihypertrophic, and anti-inflammatory effects [5e8]. Motivated by these findings, we investigated whether CNP attenuates pathology in the LPS-induced ALI model and examined the underlying mechanisms responsible for the effects of CNP.
2.
Materials and methods
2.1.
Animals
C57BL/6 mice (male, 6-wk old, weighing 18e20 g each) were purchased from Japan SLC (Shizuoka, Japan). All experimental protocols described in the present study were approved by the Animal Care Ethics Committee of the National Cerebral and Cardiovascular Center Research Institute.
2.2.
(CNP group, n ¼ 12). CNP was continuously infused using an osmotic mini-pump as previously reported [5]. The osmotic mini-pump (Alzet Model 1003D; Duret Corporation, Cupertino, CA) containing either CNP diluted in 5% glucose or vehicle was implanted subcutaneously under anesthesia in the upper back of each mouse [5]. CNP was administered at a rate of 2.5 mg/kg/min and continued until the mice were euthanized. To address the time lag between implantation and stable drug delivery [9], the pumps were implanted 24 h before LPS injection. This dose of CNP (2.5 mg/kg/min) significantly increased the blood level of cyclic guanosine monophosphate, which is the intracellular second messenger of CNP, without causing hypotension (data not shown). Mice were treated with LPS (tail vein injection, 1.0 mg/kg) 24 h before assessment, as previously described [10]. A subgroup of mice was assessed by measurements of cell counts and cytokine concentrations in bronchoalveolar lavage fluid (BALF) as follows, whereas the remainder of the mice was euthanized for histologic and gene expression analysis of the lung. The right lung was excised, frozen immediately in liquid nitrogen, and stored at 80 C for gene expression analysis. The left lung was fixed by intratracheal instillation of 4% paraformaldehyde for 24 h and subsequently embedded in paraffin, then sliced in three pieces. Paraffin sections were stained with hematoxylin-eosin (HE) for indirect immunohistochemistry for myeloperoxidase (MPO; Thermo Fisher Scientific, Waltham, MA), which is expressed in neutrophil granulocytes.
2.3.2.
Bronchoalveolar lavage technique
BALF was assessed as previously described [11e13]. In an open-chest procedure, a mouse trachea was cannulated (20gauge intravenous catheter) and 1 mL of phosphate-buffered saline (PBS) was infused intratracheally and withdrawn. This lavage technique was repeated two additional times with the same 1.0-mL solution. BALFs were centrifuged at 300g for 10 min at 4 C, and the supernatants were stored at 80 C until cytokines and albumin were measured. The cell pellet was resuspended in 0.5-mL PBS, and slides were prepared by cytocentrifugation (Cytospin 4; Thermo), and stained with Diff-Quick (Dade Behring, Dudingen, Switzerland). Cell differential counts in the BALF were determined using morphologic criteria under a light microscope; 1000 cells per slide were evaluated.
Chemicals and reagents 2.3.3.
CNP, purchased from Peptide Institute, Inc (Osaka, Japan), was dissolved in 5% glucose solution at a concentration of 20 mg/ mL. LPS from Escherichia coli O55 was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan).
2.3.
In vivo study
2.3.1.
LPS-induced ALI model
To study the effects of CNP on the lung with systemic inflammation, an animal model of ALI induced by LPS administration with or without CNP was used. This protocol resulted in the creation of three groups as follows: normal control mice (control group, n ¼ 13), LPS mice treated with vehicle (vehicle group, n ¼ 12), and LPS mice treated with CNP
Immunohistochemistry
The tissue sections were deparaffinized, and endogenous peroxidase was blocked with 3% H2O2 for 30 min. After each step, the tissue sections were rinsed twice in PBS for 5 min. The deparaffinized tissue sections were incubated with Protein Block (DakoCytomation, Glostrup, Denmark) for 15 min. The rabbit antiemouse MPO antibody was diluted in antibody diluent buffer (DakoCytomation, dilution 1:100) and applied at room temperature for 90 min. After incubation with primary antibodies, the slides were incubated with biotinylated goat antierabbit IgG (Vector Laboratories, Burlingame, CA) for 30 min, followed by incubation with a peroxidase-conjugated avidinebiotin complex (Vectastain ABC kit; Vector Laboratories) for 30 min. The sections were visualized with 0.5% diaminobenzidine (DakoCytomation) and 0.3% hydrogen
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 6 3 1 e6 3 7
peroxide, and counterstained with HE. The immunohistochemically stained sections were photographed (FSX100; Olympus, Tokyo, Japan), and 10 randomly selected fields per section and 3 sections per animal were analyzed.
2.3.4.
Gene expression analysis
Total RNA was isolated from lung tissue homogenates using guanidinium-phenol-chloroform extraction and an RNeasy mini kit (Qiagen, Hilden, Germany). The obtained RNA was reverse-transcribed into complementary DNA using a SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA). Quantitative polymerase chain reaction assays were conducted in a 96-well plate using SYBR Premix Ex Taq (Takara, Shiga, Japan) with a Light Cycler 480 System II (Roche Applied Science, Indianapolis, IN). The primers had the following sequences: E-selectin, sense 50 -GAGCTCAGAATCTACAGTGTACCTC-30 , and antisense 50 -GGATTTGTGGTGTCCACTGCCCTTG-3’; monocyte chemoattractant protein-1 (MCP-1), sense 50 -GCAGGTGTCCCAA AGAAGCTGTAGT-30 , and antisense 50 -CAGAAGTGCTTGAGGT GGTTGTGGA-3’; S100A8, sense 50 -CCATGCCCTCTACAAGAAT GAC-30 , and antisense 50 -TCTTTGTGAGATGCCACACCCA-30 , and 36B4, sense 50 -TCATTGTGGGAGCAGACAATGTGGG-30 and antisense 50 -AGGTCCTCCTTGGTGAACACAAAGC-3’. The polymerase chain reaction settings were as follows: initial
633
denaturation at 95 C was followed by 38 cycles of amplification for 5 s at 95 C/20 s at 60 C (E-selectin), 5 s at 95 C/20 s at 58 C (MCP-1), and 5 s at 95 C/10 s at 55 C/15 s at 72 C (S100A8), with subsequent melting curve analysis, increasing the temperature from 72 Ce98 C. Quantification of gene expression was calculated relative to the housekeeping gene 36B4.
2.3.5.
Cytokines measurement
The concentrations of tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), MCP-1, macrophage inflammatory protein-2 (MIP-2), and keratinocyte-derived chemokine (KC) in BALF were measured using enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol; the detection limits of the kits were 23.4, 7.8, 15.6, 7.8, and 15.6 pg/mL, respectively. The cytokine concentrations in BALF were normalized to albumin content measured using a Mouse Albumin enzyme-linked immunosorbent assay Kit (AKRAL-121; Shibayagi, Gunma, Japan).
2.4.
Statistical analysis
Data were entered into a database and analyzed using StatView for Windows (SAS Institute Inc., Cary, NC), and expressed as means standard error. Between-group
Fig. 1 e HE staining and immunohistochemical detection of MPO in murine lung. LPS-induced inflammatory cell infiltration in the lung parenchyma compared with that in control mice (A and B), whereas CNP attenuated inflammatory cell infiltration in the lung compared with that in LPS-treated mice (C). The number of MPO-positive cells in the lungs of control mice was significantly increased after LPS stimulation (DeG), whereas their number was significantly decreased in CNPtreated mice compared with that in control mice after LPS injection (F and G). The images are representative of lung specimens for each condition. Scale bar, 100 mm. Data are expressed as means ± standard error (n [ 3 in each group); *P < 0.05 versus control group; #P < 0.05 versus vehicle group.
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j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 6 3 1 e6 3 7
A
B
C
D
Fig. 2 e The number of total cells (A), macrophages (B), neutrophils (C), and lymphocytes (D) in the BALF of LPS-induced ALI in mice. After LPS administration, total cell counts and individual cell counts were significantly increased, whereas CNP pretreatment markedly reduced these cell counts compared with those in the vehicle group. Data are expressed as mean ± standard error (n [ 5e6 in each group), *P < 0.05 versus control group; #P < 0.05 versus vehicle group.
comparisons were performed using the ManneWhitney U test. For multiple-group comparisons, a one-way analysis of variance, followed by the post hoc Fisher least significant difference test, was used. P < 0.05 was considered significant.
3.
Results
3.1. Effect of CNP on pulmonary histopathologic changes in mice with ALI Lung tissues from the control group showed normal structure under light microscopy (Fig. 1A). In the vehicle group, the HEstained lungs exhibited obvious infiltration of inflammatory cells (Fig. 1B). In the CNP group, the histopathologic changes in the lung were less severe than those observed in the vehicle group, especially with respect to inflammatory cell infiltration (Fig. 1C). The number of MPO-positive cells was significantly increased in the lungs of the vehicle group compared with that
in the control group, and significantly attenuated in the lungs of the CNP group compared with that in the vehicle group (Fig. 1DeG).
3.2. Effect of CNP on cell counts and concentrations of inflammatory cytokines in BALF of mice with ALI A significant increase in the leukocyte cell counts was observed in BALF from LPS-injected mice. In BALF, both total and individual cell counts, including macrophages, neutrophils, and lymphocytes, were significantly lower in the CNP group compared with those in the vehicle group (Fig. 2AeD). The concentrations of TNF-a, MIP-2, MCP-1, IL-6 (5.9-fold; P < 0.05), and KC (1.9-fold; P < 0.05) in BALF were markedly increased 24 h after LPS administration. CNP pretreatment significantly reduced the production of TNF-a (by 18.2%; P < 0.05), MIP-2 (by 29.3%; P < 0.05), MCP-1 (by 38.1%; P < 0.05), IL-6 (by 29.5%; P < 0.05), and KC (by 71.8%; P < 0.05; Fig. 3AeE).
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A
B
D
E
635
C
Fig. 3 e Cytokine levels in BALF, including TNF-a (A), MIP-2 (B), IL-6 (C), MCP-1 (D), and KC (E). Elevated concentrations of TNF-a, MIP-2, IL-6, MCP-1, and KC in BALF after LPS administration were significantly attenuated by CNP pretreatment. Normalized cytokine levels are expressed relative to albumin content. Data are expressed as means ± standard error (n [ 5e6 in each group); *P < 0.05 versus control group; #P < 0.05 versus vehicle group. ND [ not detected.
3.3. Effect of CNP on MCP-1, S100A8, and E-selectin gene expressions in lung Increased gene expression in the lung was observed for MCP-1 (107.8-fold; P < 0.05), S100A8 (13.1-fold, P < 0.05), and
A
B
E-selectin (8.0-fold, P < 0.05) after LPS injection. CNP pretreatment significantly decreased the expressions of MCP-1 (by 44.4%, P < 0.05), S100A8 (by 64.9%, P < 0.05), and E-selectin (by 37.5%, P < 0.05) compared with those in the vehicle group (Fig. 4).
C
Fig. 4 e Real-time polymerase chain reaction analysis of MCP-1 (A), S100A8 (B), and E-selectin (C) gene expressions in murine lung. CNP pretreatment decreased the LPS-induced expression of MCP-1, S100A8, and E-selectin compared with that in the vehicle group. Data are expressed as means ± standard error (n [ 4 in each group); *P < 0.05 versus control group; #P < 0.05 versus vehicle group.
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4.
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Discussion
The present results showed that CNP has a prophylactic effect on LPS-induced pulmonary inflammation and inflammatory cell infiltration in the lung parenchyma. The present findings indicate that CNP administration may be a treatment option to prevent pulmonary inflammation during the perioperative period for pulmonary resection surgery with high-risk comorbidity, such as interstitial lung disease. The NP family consists of the structurally homologous but genetically distinct peptide hormones ANP, BNP, and CNP [14]. NPs exert their effect by activating guanylyl cyclase (GC)coupled cell surface receptors and regulate a variety of physiological processes by intracellular accumulation of their second messenger cyclic guanosine monophosphate [14]. CNP is expressed in a wide variety of tissues and acts locally as an autocrine and paracrine hormone by activating its specific receptor, GC-B [15]. Although ANP and BNP were well characterized as cardiac hormones, which bind to their specific receptor, GC-A, and have been used in clinical medicine, evidence indicating the role of CNP has been published in the last decade but has yet to be applied clinically [16]. In vivo administration of CNP improves cardiac function and attenuates cardiac remodeling after myocardial infarction in rats because of the antifibrotic and antihypertrophic effects of CNP [17]. In terms of anti-inflammatory effects, it has been reported that CNP suppressed LPS-activated murine macrophage secretion of prostaglandin E2 [18] and inhibits vascular inflammation and intimal hyperplasia in experimental vein grafts [8]. Additionally, CNP was reported to attenuate bleomycin-induced pulmonary fibrosis [5]. Thus, we investigated whether CNP attenuates LPS-induced ALI via lung parenchyma including vascular endothelial cells and blood cells. An initial inflammatory insult to the lung results in increased expression and release of proinflammatory cytokines, such as IL-6, MCP-1, and KC [19]. This results in the activation and recruitment of neutrophils into areas of the inflamed lung. Additionally, S100A8, which is a member of the S100 protein family and is expressed in macrophages and endothelial cells, plays an important role in neutrophil migration to the site of inflammation [20]. In ALI, neutrophils are the earliest immune cells to be recruited to the site of injury, and they express multiple cytotoxic products and proinflammatory cytokines, such as TNF-a and MIP-2 [21]. Our results showed that CNP treatment attenuated the expressions of IL-6, MCP-1, KC, and S100A8 in the LPS-induced inflamed lungs and consequently attenuated neutrophil infiltration and the amount of TNF-a and MIP-2 expression in BALF. This may be a result of the anti-inflammatory effect of CNP on macrophages and vascular endothelial cells, as previously reported [5,8,18]. E-selectin is expressed specifically in vascular endothelial cells [22]. Interaction of E-selectin with its ligand leads to the rolling of leukocytes on inflamed endothelial cells, which is the first step in adhesion and transmigration to surrounding tissues [22]. In this study, LPS induced increased E-selectin gene expression in the lung and was reduced by CNP pretreatment. We previously reported the prophylactic effects of ANP in an ALI model via reduction of E-selectin expression in
vascular endothelial cells [10]. Vascular endothelial cells express GC-B, which is the specific receptor of CNP, as well as GC-A, which is the ANP receptor [23]. These findings suggest that attenuation of inflammatory cell infiltration by CNP may be regulated, at least in part, by suppression of E-selectin expression in vascular endothelial cells. CNP administration was initiated 24 h before LPS administration using an osmotic mini-pump. It has been reported that osmotic pumps require at least 5 h to achieve a steady-state delivery rate, and thus there may be a significant time lag between implanting the osmotic pump and achieving sufficient blood concentration of CNP [9]. Because this time lag could influence the effects caused by CNP, we implanted the pump 24 h before LPS administration. When LPS was injected immediately after implanting the pump, no significant effect of CNP was observed (data not shown). The need to begin CNP administration with an osmotic pump before administering LPS is a limitation of this study for assessing anti-inflammatory effects of CNP in the acute phase. In clinical settings, CNP administration initiated before surgery might attenuate the inflammatory reaction induced by surgical stress, but further exploration regarding CNP administration, including bolus infusion after disease onset, is warranted. Finally, it is important to note that CNP is an endogenous peptide and is considered a safer treatment alternative than other chemical compounds; additionally, the influence of CNP on blood pressure and heart rate is very small [7,24]. Considering that inflammatory cytokines and LPS potently stimulate CNP secretion from endothelial cells [16,25], exogenous CNP administration may reinforce potential anti-inflammatory regulation by endothelial cells. However, because the detailed mechanisms of the effects of CNP remain unexplored, further studies to elucidate the specific targets affected by CNP are required.
5.
Conclusions
In conclusion, the present study is the first to show that CNP reduces LPS-induced lung injury. The mechanism of action might involve downregulation of inflammatory cytokine expression in lung parenchyma and neutrophil migration in the lung. Additional studies are warranted to determine whether these effects can be observed in other experimental models and translated into improved clinical outcomes.
Acknowledgment This work was supported in part by a grant-in-aid for Scientific Research (90580796). Author’s contributions: T.K. and T.N. contributed to the conception and design. T.K. and H.H. contributed to the analysis and interpretation. T.K., H.H., and S.I. collected the data. T.K. wrote the article. Y.S., M.I., M.O., and K.K. did the critical revision of the article. M.M. and K.K. obtaining the funding.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 6 3 1 e6 3 7
Disclosure Conflict of interest: None declared.
references
[1] Rubenfeld GD, Caldwell E, Peabody E, et al. Incidence and outcomes of acute lung injury. N Engl J Med 2005;353:1685. [2] Alam N, Park BJ, Wilton A, et al. Incidence and risk factors for lung injury after lung cancer resection. Ann Thorac Surg 2007;84:1085. discussion 91. [3] Brigham KL, Meyrick B. Endotoxin and lung injury. Am Rev Respir Dis 1986;133:913. [4] Sudoh T, Minamino N, Kangawa K, Matsuo H. C-type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain. Biochem Biophys Res Commun 1990;168:863. [5] Murakami S, Nagaya N, Itoh T, et al. C-type natriuretic peptide attenuates bleomycin-induced pulmonary fibrosis in mice. Am J Physiol Lung Cell Mol Physiol 2004;287:L1172. [6] Itoh T, Nagaya N, Murakami S, et al. C-type natriuretic peptide ameliorates monocrotaline-induced pulmonary hypertension in rats. Am J Respir Crit Care Med 2004;170: 1204. [7] Obata H, Yanagawa B, Tanaka K, et al. CNP infusion attenuates cardiac dysfunction and inflammation in myocarditis. Biochem biophysical Res Commun 2007;356:60. [8] Schachner T, Zou Y, Oberhuber A, et al. Perivascular application of C-type natriuretic peptide attenuates neointimal hyperplasia in experimental vein grafts. Eur J Cardiothorac Surg 2004;25:585. [9] Theeuwes F, Yum SI. Principles of the design and operation of generic osmotic pumps for the delivery of semisolid or liquid drug formulations. Ann Biomed Eng 1976;4:343. [10] Nojiri T, Hosoda H, Tokudome T, et al. Atrial natriuretic peptide inhibits lipopolysaccharide-induced acute lung injury. Pulm Pharmacol Ther 2014;29:24. [11] Feng X, Jia A. Protective effect of carvacrol on acute lung injury induced by lipopolysaccharide in mice. Inflammation 2014;37:1091. [12] Liu F, Sun GQ, Gao HY, et al. Angelicin regulates LPS-induced inflammation via inhibiting MAPK/NF-kappaB pathways. J Surg Res 2013;185:300.
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[13] Ogino K, Zhang R, Takahashi H, et al. Allergic airway inflammation by nasal inoculation of particulate matter (PM2.5) in NC/Nga mice. PLoS One 2014;9:e92710. [14] Langenickel TH, Buttgereit J, Pagel-Langenickel I, et al. Cardiac hypertrophy in transgenic rats expressing a dominant-negative mutant of the natriuretic peptide receptor B. Proc Natl Acad Sci U S A 2006;103:4735. [15] Suga S, Nakao K, Itoh H, et al. Endothelial production of Ctype natriuretic peptide and its marked augmentation by transforming growth factor-beta. Possible existence of “vascular natriuretic peptide system”. J Clin Invest 1992;90: 1145. [16] Del Ry S. C-type natriuretic peptide: a new cardiac mediator. Peptides 2013;40:93. [17] Soeki T, Kishimoto I, Okumura H, et al. C-type natriuretic peptide, a novel antifibrotic and antihypertrophic agent, prevents cardiac remodeling after myocardial infarction. J Am Coll Cardiol 2005;45:608. [18] Kiemer AK, Lehner MD, Hartung T, Vollmar AM. Inhibition of cyclooxygenase-2 by natriuretic peptides. Endocrinology 2002;143:846. [19] Goodman RB, Pugin J, Lee JS, Matthay MA. Cytokinemediated inflammation in acute lung injury. Cytokine Growth Factor Rev 2003;14:523. [20] Vandal K, Rouleau P, Boivin A, Ryckman C, Talbot M, Tessier PA. Blockade of S100A8 and S100A9 suppresses neutrophil migration in response to lipopolysaccharide. J Immunol 2003;171:2602. [21] Shenkar R, Abraham E. Mechanisms of lung neutrophil activation after hemorrhage or endotoxemia: roles of reactive oxygen intermediates, NF-kappa B, and cyclic AMP response element binding protein. J Immunol 1999;163:954. [22] Jubeli E, Moine L, Vergnaud-Gauduchon J, Barratt G. Eselectin as a target for drug delivery and molecular imaging. J Control Release 2012;158:194. [23] Rautureau Y, Gowers I, Wheeler-Jones CP, Baxter GF. C-type natriuretic peptide regulation of guanosine-3’,5’-cyclic monophosphate production in human endothelial cells. Auton Autacoid Pharmacol 2010;30:185. [24] Ahluwalia A, MacAllister RJ, Hobbs AJ. Vascular actions of natriuretic peptides. Cyclic GMP-dependent and -independent mechanisms. Basic Res Cardiol 2004;99:83. [25] Suga S, Itoh H, Komatsu Y, et al. Cytokine-induced C-type natriuretic peptide (CNP) secretion from vascular endothelial cellsdevidence for CNP as a novel autocrine/paracrine regulator from endothelial cells. Endocrinology 1993;133: 3038.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
C-type natriuretic peptide attenuates lipopolysaccharide-induced acute lung injury in mice Toru Kimura, MD,a,b,* Takashi Nojiri, MD, PhD,a,b Hiroshi Hosoda, MD, PhD,c Shin Ishikane, PhD,a Yasushi Shintani, MD, PhD,b Masayoshi Inoue, MD, PhD,b Mikiya Miyazato, MD, PhD,a Meinoshin Okumura, MD, PhD,b and Kenji Kangawa, PhDa a
Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, Suita-City, Osaka, Japan b Department of General Thoracic Surgery, Osaka University Graduate School of Medicine, Suita-City, Osaka, Japan c Department of Regenerative Medicine and Tissue Engineering, National Cerebral and Cardiovascular Center Research Institute, Suita-City, Osaka, Japan
article info
abstract
Article history:
Background: C-type natriuretic peptide (CNP), secreted by vascular endothelial cells, belongs
Received 30 July 2014
to a family of peptides that includes atrial and brain natriuretic peptides. CNP exhibits
Received in revised form
many vasoprotective effects against pulmonary hypertension and pulmonary fibrosis. The
15 October 2014
objective of this study was to investigate the prophylactic effects of CNP in a mouse model
Accepted 13 November 2014
of lipopolysaccharide (LPS)-induced acute lung injury (ALI).
Available online 20 November 2014
Materials and methods: C57BL/6 mice were divided into three groups as follows: normal control
Keywords:
Twenty-four hours after tail vein injection of LPS, histopathologic, gene expression, and bron-
C-type natriuretic peptide
choalveolar lavage fluid (BALF) assessments were performed on the lungs. To examine the
Acute lung injury
neutrophils in the lungs, cells positive for myeloperoxidase staining were detected by immu-
Pulmonary inflammation
nohistochemistry. BALF cytokine levels were analyzed by enzyme-linked immunosorbent
Anti-inflammation
assays. Gene expression in lung tissue was analyzed by real-time polymerase chain reaction.
mice (n ¼ 13), LPS mice treated with vehicle (n ¼ 12), and LPS mice treated with CNP (n ¼ 12).
Results: CNP significantly attenuated the elevation of leukocyte cell counts and levels of tumor necrosis factor-alpha, macrophage inflammatory protein-2, monocyte chemoattractant protein1, interleukin-6, and keratinocyte-derived chemokine in the BALF after LPS injection. Furthermore, there were significantly fewer myeloperoxidase-positive cells in lungs treated with CNP after LPS injection. In lungs of CNP-treated mice, expression of the monocyte chemoattractant protein-1, S100A8, and E-selectin genes was significantly lower than that in vehicle-treated mice. Conclusions: CNP had a protective effect on ALI induced by LPS by reducing inflammatory cell infiltration. CNP may hold promise in therapeutic strategies for ALI after pulmonary resection surgery. ª 2015 Elsevier Inc. All rights reserved.
* Corresponding author. Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita-city, Osaka 565 8565, Japan. Tel.: þ81 6 6833 5012; fax: þ81 6 6833 9865. E-mail address: [email protected] (T. Kimura). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.11.023
632
1.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 6 3 1 e6 3 7
Introduction
Acute lung injury (ALI) is a syndrome consisting of acute respiratory failure with bilateral pulmonary infiltrates associated with both pulmonary and nonpulmonary risk factors [1]. ALI is also caused by pulmonary resection surgery because of mechanical and inflammatory stress [2]. Perioperative management of ALI is therefore an important issue for pulmonary resection surgery. Because no prophylactic strategy has been established, the development of effective treatments is desirable. Although there are numerous causes of ALI, endotoxin is thought to be the most common pathogen leading to the development of ALI. Lipopolysaccharide (LPS) is derived from the cell wall of gram-negative bacteria and is known as a major factor contributing to the development of ALI, including the recruitment of inflammatory cells into the lung with subsequent increases in capillary permeability and alveolar edema [3]. C-type natriuretic peptide (CNP), a member of the natriuretic peptide (NP) family, which includes atrial and brain natriuretic peptides (ANP and BNP, respectively), was initially isolated from porcine brain as the third member of the NP family [4]. CNP is secreted by vascular endothelial cells in a wide variety of tissues and has been shown to exhibit a wide range of vasoprotective effects against pulmonary hypertension and pulmonary fibrosis, including antifibrotic, antihypertrophic, and anti-inflammatory effects [5e8]. Motivated by these findings, we investigated whether CNP attenuates pathology in the LPS-induced ALI model and examined the underlying mechanisms responsible for the effects of CNP.
2.
Materials and methods
2.1.
Animals
C57BL/6 mice (male, 6-wk old, weighing 18e20 g each) were purchased from Japan SLC (Shizuoka, Japan). All experimental protocols described in the present study were approved by the Animal Care Ethics Committee of the National Cerebral and Cardiovascular Center Research Institute.
2.2.
(CNP group, n ¼ 12). CNP was continuously infused using an osmotic mini-pump as previously reported [5]. The osmotic mini-pump (Alzet Model 1003D; Duret Corporation, Cupertino, CA) containing either CNP diluted in 5% glucose or vehicle was implanted subcutaneously under anesthesia in the upper back of each mouse [5]. CNP was administered at a rate of 2.5 mg/kg/min and continued until the mice were euthanized. To address the time lag between implantation and stable drug delivery [9], the pumps were implanted 24 h before LPS injection. This dose of CNP (2.5 mg/kg/min) significantly increased the blood level of cyclic guanosine monophosphate, which is the intracellular second messenger of CNP, without causing hypotension (data not shown). Mice were treated with LPS (tail vein injection, 1.0 mg/kg) 24 h before assessment, as previously described [10]. A subgroup of mice was assessed by measurements of cell counts and cytokine concentrations in bronchoalveolar lavage fluid (BALF) as follows, whereas the remainder of the mice was euthanized for histologic and gene expression analysis of the lung. The right lung was excised, frozen immediately in liquid nitrogen, and stored at 80 C for gene expression analysis. The left lung was fixed by intratracheal instillation of 4% paraformaldehyde for 24 h and subsequently embedded in paraffin, then sliced in three pieces. Paraffin sections were stained with hematoxylin-eosin (HE) for indirect immunohistochemistry for myeloperoxidase (MPO; Thermo Fisher Scientific, Waltham, MA), which is expressed in neutrophil granulocytes.
2.3.2.
Bronchoalveolar lavage technique
BALF was assessed as previously described [11e13]. In an open-chest procedure, a mouse trachea was cannulated (20gauge intravenous catheter) and 1 mL of phosphate-buffered saline (PBS) was infused intratracheally and withdrawn. This lavage technique was repeated two additional times with the same 1.0-mL solution. BALFs were centrifuged at 300g for 10 min at 4 C, and the supernatants were stored at 80 C until cytokines and albumin were measured. The cell pellet was resuspended in 0.5-mL PBS, and slides were prepared by cytocentrifugation (Cytospin 4; Thermo), and stained with Diff-Quick (Dade Behring, Dudingen, Switzerland). Cell differential counts in the BALF were determined using morphologic criteria under a light microscope; 1000 cells per slide were evaluated.
Chemicals and reagents 2.3.3.
CNP, purchased from Peptide Institute, Inc (Osaka, Japan), was dissolved in 5% glucose solution at a concentration of 20 mg/ mL. LPS from Escherichia coli O55 was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan).
2.3.
In vivo study
2.3.1.
LPS-induced ALI model
To study the effects of CNP on the lung with systemic inflammation, an animal model of ALI induced by LPS administration with or without CNP was used. This protocol resulted in the creation of three groups as follows: normal control mice (control group, n ¼ 13), LPS mice treated with vehicle (vehicle group, n ¼ 12), and LPS mice treated with CNP
Immunohistochemistry
The tissue sections were deparaffinized, and endogenous peroxidase was blocked with 3% H2O2 for 30 min. After each step, the tissue sections were rinsed twice in PBS for 5 min. The deparaffinized tissue sections were incubated with Protein Block (DakoCytomation, Glostrup, Denmark) for 15 min. The rabbit antiemouse MPO antibody was diluted in antibody diluent buffer (DakoCytomation, dilution 1:100) and applied at room temperature for 90 min. After incubation with primary antibodies, the slides were incubated with biotinylated goat antierabbit IgG (Vector Laboratories, Burlingame, CA) for 30 min, followed by incubation with a peroxidase-conjugated avidinebiotin complex (Vectastain ABC kit; Vector Laboratories) for 30 min. The sections were visualized with 0.5% diaminobenzidine (DakoCytomation) and 0.3% hydrogen
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peroxide, and counterstained with HE. The immunohistochemically stained sections were photographed (FSX100; Olympus, Tokyo, Japan), and 10 randomly selected fields per section and 3 sections per animal were analyzed.
2.3.4.
Gene expression analysis
Total RNA was isolated from lung tissue homogenates using guanidinium-phenol-chloroform extraction and an RNeasy mini kit (Qiagen, Hilden, Germany). The obtained RNA was reverse-transcribed into complementary DNA using a SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA). Quantitative polymerase chain reaction assays were conducted in a 96-well plate using SYBR Premix Ex Taq (Takara, Shiga, Japan) with a Light Cycler 480 System II (Roche Applied Science, Indianapolis, IN). The primers had the following sequences: E-selectin, sense 50 -GAGCTCAGAATCTACAGTGTACCTC-30 , and antisense 50 -GGATTTGTGGTGTCCACTGCCCTTG-3’; monocyte chemoattractant protein-1 (MCP-1), sense 50 -GCAGGTGTCCCAA AGAAGCTGTAGT-30 , and antisense 50 -CAGAAGTGCTTGAGGT GGTTGTGGA-3’; S100A8, sense 50 -CCATGCCCTCTACAAGAAT GAC-30 , and antisense 50 -TCTTTGTGAGATGCCACACCCA-30 , and 36B4, sense 50 -TCATTGTGGGAGCAGACAATGTGGG-30 and antisense 50 -AGGTCCTCCTTGGTGAACACAAAGC-3’. The polymerase chain reaction settings were as follows: initial
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denaturation at 95 C was followed by 38 cycles of amplification for 5 s at 95 C/20 s at 60 C (E-selectin), 5 s at 95 C/20 s at 58 C (MCP-1), and 5 s at 95 C/10 s at 55 C/15 s at 72 C (S100A8), with subsequent melting curve analysis, increasing the temperature from 72 Ce98 C. Quantification of gene expression was calculated relative to the housekeeping gene 36B4.
2.3.5.
Cytokines measurement
The concentrations of tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), MCP-1, macrophage inflammatory protein-2 (MIP-2), and keratinocyte-derived chemokine (KC) in BALF were measured using enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol; the detection limits of the kits were 23.4, 7.8, 15.6, 7.8, and 15.6 pg/mL, respectively. The cytokine concentrations in BALF were normalized to albumin content measured using a Mouse Albumin enzyme-linked immunosorbent assay Kit (AKRAL-121; Shibayagi, Gunma, Japan).
2.4.
Statistical analysis
Data were entered into a database and analyzed using StatView for Windows (SAS Institute Inc., Cary, NC), and expressed as means standard error. Between-group
Fig. 1 e HE staining and immunohistochemical detection of MPO in murine lung. LPS-induced inflammatory cell infiltration in the lung parenchyma compared with that in control mice (A and B), whereas CNP attenuated inflammatory cell infiltration in the lung compared with that in LPS-treated mice (C). The number of MPO-positive cells in the lungs of control mice was significantly increased after LPS stimulation (DeG), whereas their number was significantly decreased in CNPtreated mice compared with that in control mice after LPS injection (F and G). The images are representative of lung specimens for each condition. Scale bar, 100 mm. Data are expressed as means ± standard error (n [ 3 in each group); *P < 0.05 versus control group; #P < 0.05 versus vehicle group.
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A
B
C
D
Fig. 2 e The number of total cells (A), macrophages (B), neutrophils (C), and lymphocytes (D) in the BALF of LPS-induced ALI in mice. After LPS administration, total cell counts and individual cell counts were significantly increased, whereas CNP pretreatment markedly reduced these cell counts compared with those in the vehicle group. Data are expressed as mean ± standard error (n [ 5e6 in each group), *P < 0.05 versus control group; #P < 0.05 versus vehicle group.
comparisons were performed using the ManneWhitney U test. For multiple-group comparisons, a one-way analysis of variance, followed by the post hoc Fisher least significant difference test, was used. P < 0.05 was considered significant.
3.
Results
3.1. Effect of CNP on pulmonary histopathologic changes in mice with ALI Lung tissues from the control group showed normal structure under light microscopy (Fig. 1A). In the vehicle group, the HEstained lungs exhibited obvious infiltration of inflammatory cells (Fig. 1B). In the CNP group, the histopathologic changes in the lung were less severe than those observed in the vehicle group, especially with respect to inflammatory cell infiltration (Fig. 1C). The number of MPO-positive cells was significantly increased in the lungs of the vehicle group compared with that
in the control group, and significantly attenuated in the lungs of the CNP group compared with that in the vehicle group (Fig. 1DeG).
3.2. Effect of CNP on cell counts and concentrations of inflammatory cytokines in BALF of mice with ALI A significant increase in the leukocyte cell counts was observed in BALF from LPS-injected mice. In BALF, both total and individual cell counts, including macrophages, neutrophils, and lymphocytes, were significantly lower in the CNP group compared with those in the vehicle group (Fig. 2AeD). The concentrations of TNF-a, MIP-2, MCP-1, IL-6 (5.9-fold; P < 0.05), and KC (1.9-fold; P < 0.05) in BALF were markedly increased 24 h after LPS administration. CNP pretreatment significantly reduced the production of TNF-a (by 18.2%; P < 0.05), MIP-2 (by 29.3%; P < 0.05), MCP-1 (by 38.1%; P < 0.05), IL-6 (by 29.5%; P < 0.05), and KC (by 71.8%; P < 0.05; Fig. 3AeE).
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A
B
D
E
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C
Fig. 3 e Cytokine levels in BALF, including TNF-a (A), MIP-2 (B), IL-6 (C), MCP-1 (D), and KC (E). Elevated concentrations of TNF-a, MIP-2, IL-6, MCP-1, and KC in BALF after LPS administration were significantly attenuated by CNP pretreatment. Normalized cytokine levels are expressed relative to albumin content. Data are expressed as means ± standard error (n [ 5e6 in each group); *P < 0.05 versus control group; #P < 0.05 versus vehicle group. ND [ not detected.
3.3. Effect of CNP on MCP-1, S100A8, and E-selectin gene expressions in lung Increased gene expression in the lung was observed for MCP-1 (107.8-fold; P < 0.05), S100A8 (13.1-fold, P < 0.05), and
A
B
E-selectin (8.0-fold, P < 0.05) after LPS injection. CNP pretreatment significantly decreased the expressions of MCP-1 (by 44.4%, P < 0.05), S100A8 (by 64.9%, P < 0.05), and E-selectin (by 37.5%, P < 0.05) compared with those in the vehicle group (Fig. 4).
C
Fig. 4 e Real-time polymerase chain reaction analysis of MCP-1 (A), S100A8 (B), and E-selectin (C) gene expressions in murine lung. CNP pretreatment decreased the LPS-induced expression of MCP-1, S100A8, and E-selectin compared with that in the vehicle group. Data are expressed as means ± standard error (n [ 4 in each group); *P < 0.05 versus control group; #P < 0.05 versus vehicle group.
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4.
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Discussion
The present results showed that CNP has a prophylactic effect on LPS-induced pulmonary inflammation and inflammatory cell infiltration in the lung parenchyma. The present findings indicate that CNP administration may be a treatment option to prevent pulmonary inflammation during the perioperative period for pulmonary resection surgery with high-risk comorbidity, such as interstitial lung disease. The NP family consists of the structurally homologous but genetically distinct peptide hormones ANP, BNP, and CNP [14]. NPs exert their effect by activating guanylyl cyclase (GC)coupled cell surface receptors and regulate a variety of physiological processes by intracellular accumulation of their second messenger cyclic guanosine monophosphate [14]. CNP is expressed in a wide variety of tissues and acts locally as an autocrine and paracrine hormone by activating its specific receptor, GC-B [15]. Although ANP and BNP were well characterized as cardiac hormones, which bind to their specific receptor, GC-A, and have been used in clinical medicine, evidence indicating the role of CNP has been published in the last decade but has yet to be applied clinically [16]. In vivo administration of CNP improves cardiac function and attenuates cardiac remodeling after myocardial infarction in rats because of the antifibrotic and antihypertrophic effects of CNP [17]. In terms of anti-inflammatory effects, it has been reported that CNP suppressed LPS-activated murine macrophage secretion of prostaglandin E2 [18] and inhibits vascular inflammation and intimal hyperplasia in experimental vein grafts [8]. Additionally, CNP was reported to attenuate bleomycin-induced pulmonary fibrosis [5]. Thus, we investigated whether CNP attenuates LPS-induced ALI via lung parenchyma including vascular endothelial cells and blood cells. An initial inflammatory insult to the lung results in increased expression and release of proinflammatory cytokines, such as IL-6, MCP-1, and KC [19]. This results in the activation and recruitment of neutrophils into areas of the inflamed lung. Additionally, S100A8, which is a member of the S100 protein family and is expressed in macrophages and endothelial cells, plays an important role in neutrophil migration to the site of inflammation [20]. In ALI, neutrophils are the earliest immune cells to be recruited to the site of injury, and they express multiple cytotoxic products and proinflammatory cytokines, such as TNF-a and MIP-2 [21]. Our results showed that CNP treatment attenuated the expressions of IL-6, MCP-1, KC, and S100A8 in the LPS-induced inflamed lungs and consequently attenuated neutrophil infiltration and the amount of TNF-a and MIP-2 expression in BALF. This may be a result of the anti-inflammatory effect of CNP on macrophages and vascular endothelial cells, as previously reported [5,8,18]. E-selectin is expressed specifically in vascular endothelial cells [22]. Interaction of E-selectin with its ligand leads to the rolling of leukocytes on inflamed endothelial cells, which is the first step in adhesion and transmigration to surrounding tissues [22]. In this study, LPS induced increased E-selectin gene expression in the lung and was reduced by CNP pretreatment. We previously reported the prophylactic effects of ANP in an ALI model via reduction of E-selectin expression in
vascular endothelial cells [10]. Vascular endothelial cells express GC-B, which is the specific receptor of CNP, as well as GC-A, which is the ANP receptor [23]. These findings suggest that attenuation of inflammatory cell infiltration by CNP may be regulated, at least in part, by suppression of E-selectin expression in vascular endothelial cells. CNP administration was initiated 24 h before LPS administration using an osmotic mini-pump. It has been reported that osmotic pumps require at least 5 h to achieve a steady-state delivery rate, and thus there may be a significant time lag between implanting the osmotic pump and achieving sufficient blood concentration of CNP [9]. Because this time lag could influence the effects caused by CNP, we implanted the pump 24 h before LPS administration. When LPS was injected immediately after implanting the pump, no significant effect of CNP was observed (data not shown). The need to begin CNP administration with an osmotic pump before administering LPS is a limitation of this study for assessing anti-inflammatory effects of CNP in the acute phase. In clinical settings, CNP administration initiated before surgery might attenuate the inflammatory reaction induced by surgical stress, but further exploration regarding CNP administration, including bolus infusion after disease onset, is warranted. Finally, it is important to note that CNP is an endogenous peptide and is considered a safer treatment alternative than other chemical compounds; additionally, the influence of CNP on blood pressure and heart rate is very small [7,24]. Considering that inflammatory cytokines and LPS potently stimulate CNP secretion from endothelial cells [16,25], exogenous CNP administration may reinforce potential anti-inflammatory regulation by endothelial cells. However, because the detailed mechanisms of the effects of CNP remain unexplored, further studies to elucidate the specific targets affected by CNP are required.
5.
Conclusions
In conclusion, the present study is the first to show that CNP reduces LPS-induced lung injury. The mechanism of action might involve downregulation of inflammatory cytokine expression in lung parenchyma and neutrophil migration in the lung. Additional studies are warranted to determine whether these effects can be observed in other experimental models and translated into improved clinical outcomes.
Acknowledgment This work was supported in part by a grant-in-aid for Scientific Research (90580796). Author’s contributions: T.K. and T.N. contributed to the conception and design. T.K. and H.H. contributed to the analysis and interpretation. T.K., H.H., and S.I. collected the data. T.K. wrote the article. Y.S., M.I., M.O., and K.K. did the critical revision of the article. M.M. and K.K. obtaining the funding.
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Disclosure Conflict of interest: None declared.
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