http://informahealthcare.com/iht ISSN: 0895-8378 (print), 1091-7691 (electronic) Inhal Toxicol, 2014; 26(3): 185–192 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/08958378.2013.872213
RESEARCH ARTICLE
Adenovirus-delivered angiopoietin-1 suppresses NF-kB and p38 MAPK and attenuates inflammatory responses in phosgene-induced acute lung injury Dai-Kun He1,2, Yi-Ru Shao1,2, Lin Zhang1,2, Jie Shen1,2, Zhi-Yue Zhong1,2, Jing Wang1,2, and Guoxiong Xu3 1
Center of Emergency & Intensive Care Unit, 2Medical Center of Chemical Injury, and 3Center Laboratory, Jinshan Hospital, Fudan University, Shanghai, P.R. China Abstract
Keywords
Animals exposed to phosgene (Psg) result in acute lung injury (ALI). We have recently reported that angiopoietin-1 (Ang1) reduces inflammation and vascular hyperpermeability in ALI animals. In this study, we examined whether the beneficial effects of adenovirus-delivered Ang1 (Ad/Ang1) on inflammatory responses in Psg-induced ALI rats are due to the suppression of the nuclear factor-kappa B (NF-kB) and p38 mitogen-activated protein kinase (MAPK) pathways, which play crucial roles in inflammatory responses in ALI. We demonstrated that Psg increased Ang2 and inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukin (IL)-4 (IL-4), IL-6, IL-8, and IL-10, in the serum and bronchoalveolar lavage fluid of ALI rats, determined by ELISA. Ang1 inhibits pro-inflammatory mediators (TNF-a, IL-6 and IL-8) and has no effect on anti-inflammatory mediators (IL-4 and IL-10). Furthermore, the inhibitory action of Ang1 was mediated by the suppression of the NF-kB and p38 MAPK pathways, leading to the attenuation of inflammatory responses of ALI. Thus, Ad/Ang1 may provide a useful tool for the effective treatment in Psg-induced ALI.
Acute lung injury, angiopoietin-1, cytokine, NF-kB, p38 MAPK, phosgene
Phosgene (Psg) is being used as a common industrial chemical in manufacturing dyestuff, plastics, pesticides and rubbers. Psg is usually inhaled due to its accidential exposure. Our previous studies have shown that the inhalation of Psg leads to acute lung injury (ALI) in rats (Shen et al., 2012, 2013). It has also been reported that short-time exposure of Psg causes ALI, whereas prolong exposure leads to fatal acute respiratory distress syndrome (ARDS) (Sciuto, 1998; Sciuto et al., 1996). ALI and ARDS are life-threatening syndromes characterized by inflammation and increased vascular permeability (Wheeler & Bernard, 2007). The excessive liberation of pro-inflammatory cytokines and mediators in circulation during ALI/ARDS processes instigates and intensifies the pulmonary inflammatory cascade (Dechert et al., 2012; van der Heijden et al., 2009). Among these inflammatory factors, tumor necrosis factor-a (TNF-a), interleukin (IL)-4 (IL-4), IL-6, IL-8 and IL-10 are involved in cellular events and play key roles in pulmonary injury
Address for correspondence: Guoxiong Xu, MD, PhD, Center Laboratory, Jinshan Hospital, Fudan University, 1508 Longhang Road, Shanghai 201508, P.R. China. Tel: +86-21-34189990. Fax: +86-2167226910. E-mail: [email protected] Jie Shen, MD, Center of Emergency & Intensive Care Unit, Jinshan Hospital, Fudan University, 1508 Longhang Road, Shanghai 201508, P.R. China. Tel: +86-18930819779. Fax: +86-21-67226910. E-mail: [email protected]
Received 19 October 2013 Revised 28 November 2013 Accepted 2 December 2013 Published online 31 January 2014
(Bhatia & Moochhala, 2004; Hodge et al., 2002; Huaux et al., 2003; Lucas et al., 2009; Ware, 2005). It is well documented that the pro-inflammatory and antiinflammatory cytokines are mediated by the p38 mitogenactivated protein kinase (MAPK) and nuclear factor-kappa B (NF-kB) pathways. There are three MAPKs: extracellular signal-regulated kinases (ERK), c-Jun NH2-terminal kinases (JNK) and p38 MAPK (Johnson & Lapadat, 2002; Pearson et al., 2001). Among them, p38 MAPK regulates many cytokines in ALI/ARDS caused by various reasons (Arcaroli et al., 2001; Tamura et al., 1998). The activation of p38 MAPK signaling leads to the activation of NF-kB, a transcription factor, by modulating the phosphorylation of p65 NF-kB (Kweon et al., 2006), which plays a central role in the onset of inflammation and increases the level of inflammatory mediators (Park & Christman, 2006). Indeed, the suppression of the MAPK and NF-kB pathways contributes to the attenuation of inflammatory responses in ALI (Chi et al., 2013). The angiopoietin-Tie2 system is involved in the regulation of inflammation, hyperpermeability, vasoreactive stimuli and apoptosis in the endothelium (van der Heijden et al., 2009). Angiopoietin-1 (Ang1) reduces inflammation and vascular hyperpermeability, whereas angiopoietin-2 (Ang2) has an opposite effect on inflammation (Sandhu et al., 2004; Thurston et al., 2000). Both Ang1 and Ang2 are ligands for the receptor tyrosine kinase Tie2 (Tsigkos et al., 2003). However, Ang2 is able to antagonize the elevated Ang1-activation (Hansen et al.,
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History
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2010). Ang1 functionally binds to and stimulates monocytes via p38, which is associated with pro-inflammatory cytokine TNF-a (Seok et al., 2013). Our previous study demonstrated that adenovirus-delivered Ang1 (Ad/Ang1) has beneficial effects on Psg-induced lung injury though it reduces endothelial permeability and inflammation in vivo (Shen et al., 2013). However, whether Ang1 inhibits Ang2 and whether Ang1 depresses the influence of the p38 MAPK pathway on TNF-a-induced NF-kB activation in Psg-induced ALI have not been investigated. In this study, we examined the relationship between Ang1 and inflammatory responses and studied the suppressive effects of Ad/Ang1 on NF-kB activation and phosphorylation of p38 (p-p38) associated with inflammatory responses in Psg-induced ALI.
Materials and methods Animals Sixty male Sprague-Dawley (SD) rats were used for the study and the experiment designs were the same as in our previous study (Shen et al., 2013). We randomly divided the animals into six groups: control (Air), adenovirus control (Air + Ad), adenovirus-Ang1 (Air + Ad/Ang1), Psg gas alone, Psg plus adenovirus control (Psg + Ad) and Psg plus adenovirus-Ang1 (Psg + Ad/Ang1). At least three animals were used in each group. The left lung from each rat was collected for bronchoalveolar lavage fluid (BALF), RNA or protein extraction. This work was approved by the Ethic Committee of Jinshan Hospital for Animal Care, according to the Guidelines provided by Chinese Academy of Medical Sciences.
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an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer’s instructions. RNA extraction and quantitative real-time RT-PCR Total RNA was extracted and real-time RT-PCR was performed as described previously. The primer sequences were as follow: rat Ang2 50 -GTCAACACCTTTATCCATGA CACCA-30 (forward) and 50 -GCAAGTGGTGACCTGGAAG TGAG-30 (reverse), rat NF-kB 50 –GGAGTACGACAACATC TCGTTGG-30 (forward) and 50 -TGTAGAGGTGTCGTCCCA TCGT-30 (reverse), rat p38 50 -ACCACGACCCTGATGATG AGC-30 (forward) and 50 -GGTCAGGCTCTTCCATTCGTC T-30 (reverse), rat GAPDH 50 -CCTCAAGATTGTCAGCAA T-30 (forward) and 50 -CCATCCAGCAGTCTTCTGAGT-30 (reverse). All experiments were conducted in triplicate and repeated three times. Western blot The lung tissues were homogenized in RIPA lysis buffer and equal amount of total protein was subjected to SDSPAGE and transferred to PVDF membranes as described previously (Shen et al., 2013). Anti-Ang2, anti-p38, antiphospho-p38, anti-NF-kB p65 and anti-GAPDH were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The primary antibody was incubated overnight at 4 C. The secondary antibody (anti-rabbit or anti-mouse HRPconjugated IgG) was incubated for one hour at room temperature. The specific bands were visualized using enhanced chemiluminescence reagents (GE Healthcare, Biosciences, Pittsburgh, PA).
Exposure to Psg Psg gas was prepared as described previously (Shen et al., 2013). The animals were exposed to the gas at a final concentration of 8.33 g/m3 in an enclosed cabinet for 5 min, placed in separate cabinets with fresh air and then sacrificed after 36 hours of Psg exposure. Treatment with Ad/Ang1 Ang1-overexpressing rats were established by an intravenous injection of Ad/Ang1 at an optimized dose of 1 108 pfu/ml via tail vein an hour post-Psg exposure as described previously (Shen et al., 2013). The animals were sacrificed after 36 hours of administration. Blood and BALF sample collection and lung tissue preparation The blood was collected from abdominal aorta 36 hours postPsg inhalation, and the BALF was collected from the left lung. The serum and BALF were used to measure the concentrations of Ang2 and inflammatory cytokines. The right lung was perfused with saline and used for RT-PCR and Western Blot analyses. Enzyme-linked immunosorbent assay The concentrations of Ang2, TNF-a, IL-4, IL-6, IL-8, and IL-10 in the serum and BALF were measured using
Statistical analyses All statistical analyses were carried out as described previously (Shen et al., 2013). The differences were considered significant at values of p50.05.
Results Reduction of Psg-induced Ang2 expression by Ad/Ang1 Following our previous work (Shen et al., 2013), we successfully replicated the model of Psg-induced ALI in rats, as demonstrating the overexpression of Ad/Ang1 in the lung detected by fluorescent microscopy and Western blot (data now shown). Since ALI is associated with a decrease in Ang1 and an increase in Ang2, we examined whether the overexpression of Ad/Ang1 reverses Psg-induced upregulation of Ang2 in ALI. We found that the level of Ang2 in the serum and BALF was significantly elevated after Psg exposure detected by ELISA (Figure 1A and B). However, this Psg-induced increase in Ang2 was reduced after Ad/Ang1 treatment. Up-regulation of Ang2 expression in the lungs was also detected at both mRNA and protein levels as assessed by qRT-PCR (Figure 1C) and Western blot (Figure 1D and E), respectively. This Psg-induced upregulation of Ang2 in ALI was significantly deceased after administration of Ad/Ang1.
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Figure 1. Detection of Ang2 in air- and Psg-exposed rats. The concentration of Ang2 in the serum (A) and BALF (B) was measured by ELISA. The Ang2 mRNA expression was determined by qRT-PCR (C) and the Ang2 protein expression was determined by Western blot (D, E). Data represent mean ± SEM. Different letters denote significance (p50.05, n ¼ 3 experiments). Air, air-exposed control; Air + Ad, air-exposed animal with adenovirus alone; Air + Ad/Ang1, air-exposed animal with adenovirus-delivered Ang1; Psg, Psg-exposed animal; Psg + Ad, Psg-exposed animal with adenovirus alone; Psg + Ad/Ang1, Psg-exposed animal with adenovirus-delivered Ang1.
Secreted inflammatory cytokines in the serum and BALF Pro-inflammatory cytokines TNF-a, IL-6 and IL-8 are critical factors and their concentrations are known to be altered in the process of ALI. Subsequently, we examine whether the levels of secreted pro-inflammatory cytokines in the serum and BALF are altered after Psg exposure. We found that secreted TNF-a, IL-6 and IL-8 levels, detected by ELISA, were significantly increased in the serum (Figure 2A, C and E) and BALF (Figure 2B, D and F) of Psg-exposed rats as compared with air-exposed rats. Interestingly, the increase in the levels of TNF-a, IL-6, and IL-8 after Psg exposure was significantly reversed after the treatment of Ad/Ang1 (Figure 2). It has been shown that IL-4 and IL-10 are critical anti-inflammatory cytokines during ALI (Dinarello, 2000).
Here we found that secreted IL-4 and IL-10 were also increased in the serum (Figure 3A and C) and BALF (Figure 3B and D) of Psg-exposed rats as compared with air-exposed rats. Though Ad/Ang1 reversed pro-inflammatory cytokines in Psg-induced ALI, statistical analysis showed that IL-4, which was upregulated after Psg exposure, was not significantly affected by Ad/Ang1 in the serum and BALF (Figure 3A and B). However, IL-10, which was upregulated after Psg exposure, was partially, but not significantly, reversed after the treatment of Ad/Ang1 in the serum and BALF (Figure 3C and D). Effect of Ad/Ang1 on the expression of p38 mRNA and protein in Psg-exposed rats The expression of p38 mRNA was not altered in the lungs of Psg-induced ALI rats (Figure 4A). The administration
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Figure 2. Measurement of secreted TNF-a, IL-6 and IL-8 levels in the serum and BALF by ELISA. The secreted TNF-a levels were detected in the serum (A) and BALF (B). The secreted IL-6 levels were detected in the serum (C) and BALF (D). The secreted IL-8 concentration was detected in rat serum (E) and BALF (F). Data represent mean ± SEM. Different letters denote significance (p50.05, n ¼ 3 experiments). Air, air-exposed control; Air + Ad, air-exposed animal with adenovirus alone; Air + Ad/Ang1, air-exposed animal with adenovirus-delivered Ang1; Psg, Psg-exposed animal; Psg + Ad, Psg-exposed animal with adenovirus alone; Psg + Ad/Ang1, Psg-exposed animal with adenovirus-delivered Ang1.
of Ad and Ad/Ang1 did not change the p38 mRNA expression (Figure 4A). By comparing total p38 protein expression between all the tested groups, we found that the expression of total p38 protein was quite stable. There was no significant difference in total p38 protein expression after Psg exposure with or without Ad/Ang1 treatment, compared with airexposed control (Figure 4B and C). However, we observed that phospho-p38 protein was significantly increased after Psg exposure. The increased phosphor-p38 in the Psg-exposed rats was partially, but significantly, reversed after Ad/Ang1 treatment (Figure 4B and D).
Effect of Ad/Ang1 on NF-kB mRNA and protein expression in Psg-exposed rats Next, we examined whether Ad/Ang1 affects the expression of p65 NF-kB. We found that NF-kB mRNA expression was significantly increased in Psg-expose rats compared with airexposed rats (Figure 5A). The administration of Ad/Ang1 significantly reduced Psg-induced NF-kB mRNA expression. We also observed that NF-kB protein expression increased after Psg exposure (Figure 5B and C). The increase in NF-kB protein expression in the Psg-exposed rats revised after
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Figure 3. Measurement of secreted IL-4 and IL-10 levels in the serum and BALF by ELISA. The secreted IL-4 levels were detected in the serum (A) and BALF (B). The secreted IL-10 levels were detected in the serum (C) and BALF (D). Data represent mean ± SEM. Different letters denote significance (p50.05, n ¼ 3 experiments). Air, air-exposed control; Air + Ad, air-exposed animal with adenovirus alone; Air + Ad/Ang1, air-exposed animal with adenovirus-delivered Ang1; Psg, Psg-exposed animal; Psg + Ad, Psg-exposed animal with adenovirus alone; Psg + Ad/Ang1, Psg-exposed animal with adenovirus-delivered Ang1.
Ad/Ang1 treatment (Figure 5B and C). Ad alone did not affect the expression of NF-kB.
Discussion In our previous study using Psg-induced ALI model, we demonstrated that Ad/Ang1 has a protective benefit which prevents the damage of the lung by stabilizing vascular endothelial growth factor (VEGF), suppressing leukocytes infiltration and reducing inflammation (Shen et al., 2013). In the present study, we successfully replicated the model of Psg-induced ALI in rats and examined the inflammatory cytokines in the serum and BALF with or without Ad/Ang1 treatment. The administration of Ad/Ang1 resulted in partial reversion of the adverse effects of Psg in ALI, especially for the inflammatory cytokines. We have also explored the effect of Ad/Ang1 on the regulation of NF-kB and the activation of p38 MAPK, both of which play important roles in regulating the inflammatory cytokines. Currently, there is no specific and effective treatment available for ALI. Adenoviral gene delivery as gene therapy has been used in several animal models (Jenkins et al., 2003; Suzuki et al., 2001). Previous reports from us and others showed that Ad/Ang1 inhibits the progression of the damage in ALI after Psg inhalation (Shen et al., 2013) and lipopolysaccharide instillation (Xu et al., 2011). It has been shown that
Ang1 and Ang2 are competitive binding to the tyrosine kinase receptor Tie2 (Tsigkos et al., 2003). Ang2 blocks the effects of Ang1 and antagonizes Tie2 (Hansen et al., 2010). Our previous and current studies show that Psg is an important modulator of the angiopoietin family by inhibiting Ang1 and Tie2 and stimulating the expression of Ang2. These results support the fact that angiopoietin/Tie2 system is important in mediating the pulmonary vascular and inflammatory responses. Current data confirmed the previous report (Sciuto et al., 2003) that TNF-a, IL-6 and IL-8 in the serum and BALF were significantly higher in Psg-exposed mice than airexposed controls. Time-course study showed that IL-10 was significantly higher in Psg-exposed, whereas IL-4 was significantly lower in the Psg-exposed mice than in the airexposed mice from 4 to 8 hours post-exposure (Sciuto et al., 2003). In our rat model, however, we found that both IL-4 and IL-10 were significantly increased at 36 hours post-Psg exposure. The treatment of Ad/Ang1 significantly decreased serum and BALF levels of TNF-a, Il-6 and IL-8, respectively, not IL-4 and IL-10, leading the speculation that Ad/Ang1 decreases pro-inflammatory cytokines, not anti-inflammatory cytokines, to reduce lung permeability and inflammation responses. The future study is needed to assess the temporal changes of pro- and anti-inflammatory markers in Psginduced ALI animals after Ad/Ang1 treatment.
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Figure 4. Expression of p38 mRNA and protein in the lung. (A) mRNA expression of p38 detected by qPCR. (B) Western blot detection for p38 protein. (C) Semi-quantification of total p38 protein in Western blot. (D) Semi-quantification of phosphorylated p38 protein in Western blot. Data represent mean ± SEM. Different letters denote significance (p50.05, n ¼ 3 experiments). Air, air-exposed control; Air + Ad, air-exposed animal with adenovirus alone; Air + Ad/Ang1, air-exposed animal with adenovirus-delivered Ang1; Psg, Psg-exposed animal; Psg + Ad, Psg-exposed animal with adenovirus alone; Psg + Ad/Ang1, Psg-exposed animal with adenovirus-delivered Ang1.
Previous studies found that the inhibition of the NF-kB and p38 MAPK pathways reduces TNF-a-stimulated IL-8 expression (Everhart et al., 2006; Han et al., 2009; Kuldo et al., 2005; Su et al., 2008). Ang1, as opposed to Ang2, promotes neutrophil IL-8 synthesis and release through the activation of the p42/44 MAPK pathway (Neagoe et al., 2012). NF-kB is a family of DNA-binding proteins that is required for the transcription of many pro-inflammatory mediators and plays an important role in the pathogenesis of ALI (Kim et al., 2006; Liu et al., 2008). The activation of NF-kB leads to coordinated expression of various mediators of inflammation and perpetuation of the inflammatory response (Oeckinghaus & Ghosh, 2009; Park & Christman, 2006). It has been shown that NF-kB activation correlates with severity of lung injury. Suppression in the NF-kB pathway is beneficial even after the onset of lung inflammation (Everhart et al., 2006) and, therefore, NF-kB is an obvious target for anti-inflammatory treatment (Aghai et al., 2006). Thus, downregulation of NF-kB activity is a useful strategy to regulate inflammatory response. Our studies indicated that the expression of NF-kB factor p65 increased in Psg-induced ALI. The increase in NF-kB may lead to an increase in pro-inflammatory cytokines. Administration of Ad/Ang1 decreases Psg-induced
NF-kB activation, which in turn, inhibits the production of TNF-a, IL-6 and IL-8. This result suggests that the suppression of the NF-kB signaling pathway to reduce the production of inflammatory mediators is one of the mechanisms of Ad/Ang1 therapy. Previous studies showed that the activation of p38 MAPK is important in inflammatory mediator induction and involved in the ALI process (Kaminska et al., 2009). For example, the p38 MAPK pathway is critically important in regulating IL-8 expression in HUVECs (Kuldo et al., 2005) and enhances the production of IL-4 and IL-10 (Yeh et al., 2007). Current study showed that Psg activated the p38 MAPK pathway by the increase of p38 phosphorylation rather than by the regulation of total p38 in the lungs. Administration of Ad/Ang1 markedly inhibited Psg-induced phosphorylation of p38. These results indicate that the effects of Ang1 on the production of pro-inflammatory mediators may occur by blocking the p38 MAPK signaling pathway. In conclusion, we have demonstrated that Ang1 inhibits pro-inflammatory mediators, such as TNF-a, IL-6 and IL-8, and has no effect on anti-inflammatory mediators, such as IL-4 and IL-10, in the serum and BALF of ALI rats. This inhibitory action of Ang1 was mediated by the suppression of
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the NF-kB and p38 MAPK pathways, leading to the attenuation of inflammatory responses. Thus, Ad/Ang1 may provide a useful tool for the effective treatment in Psginduced ALI.
Declaration of interest The authors report no declarations of interest. This research was supported by National Youth Nature Science Foundation of China (No. 81101412) to DKH and a start-up fund of research from Jinshan Hospital (2012-2) to GX.
References
Figure 5. Expression of NF-kB mRNA and protein in the lung. (A) mRNA expression of NF-kB by qPCR. (B) Western blot detection for NF-kB protein. (C) Semi-quantification of NF-kB protein in Western blot. Data represent mean ± SEM. Different letters denote significance (p50.05, n ¼ 3 experiments). Air, air-exposed control; Air + Ad, airexposed animal with adenovirus alone; Air + Ad/Ang1, air-exposed animal with adenovirus-delivered Ang1; Psg, Psg-exposed animal; Psg + Ad, Psg-exposed animal with adenovirus alone; Psg + Ad/Ang1, Psg-exposed animal with adenovirus-delivered Ang1.
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RESEARCH ARTICLE
Adenovirus-delivered angiopoietin-1 suppresses NF-kB and p38 MAPK and attenuates inflammatory responses in phosgene-induced acute lung injury Dai-Kun He1,2, Yi-Ru Shao1,2, Lin Zhang1,2, Jie Shen1,2, Zhi-Yue Zhong1,2, Jing Wang1,2, and Guoxiong Xu3 1
Center of Emergency & Intensive Care Unit, 2Medical Center of Chemical Injury, and 3Center Laboratory, Jinshan Hospital, Fudan University, Shanghai, P.R. China Abstract
Keywords
Animals exposed to phosgene (Psg) result in acute lung injury (ALI). We have recently reported that angiopoietin-1 (Ang1) reduces inflammation and vascular hyperpermeability in ALI animals. In this study, we examined whether the beneficial effects of adenovirus-delivered Ang1 (Ad/Ang1) on inflammatory responses in Psg-induced ALI rats are due to the suppression of the nuclear factor-kappa B (NF-kB) and p38 mitogen-activated protein kinase (MAPK) pathways, which play crucial roles in inflammatory responses in ALI. We demonstrated that Psg increased Ang2 and inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukin (IL)-4 (IL-4), IL-6, IL-8, and IL-10, in the serum and bronchoalveolar lavage fluid of ALI rats, determined by ELISA. Ang1 inhibits pro-inflammatory mediators (TNF-a, IL-6 and IL-8) and has no effect on anti-inflammatory mediators (IL-4 and IL-10). Furthermore, the inhibitory action of Ang1 was mediated by the suppression of the NF-kB and p38 MAPK pathways, leading to the attenuation of inflammatory responses of ALI. Thus, Ad/Ang1 may provide a useful tool for the effective treatment in Psg-induced ALI.
Acute lung injury, angiopoietin-1, cytokine, NF-kB, p38 MAPK, phosgene
Phosgene (Psg) is being used as a common industrial chemical in manufacturing dyestuff, plastics, pesticides and rubbers. Psg is usually inhaled due to its accidential exposure. Our previous studies have shown that the inhalation of Psg leads to acute lung injury (ALI) in rats (Shen et al., 2012, 2013). It has also been reported that short-time exposure of Psg causes ALI, whereas prolong exposure leads to fatal acute respiratory distress syndrome (ARDS) (Sciuto, 1998; Sciuto et al., 1996). ALI and ARDS are life-threatening syndromes characterized by inflammation and increased vascular permeability (Wheeler & Bernard, 2007). The excessive liberation of pro-inflammatory cytokines and mediators in circulation during ALI/ARDS processes instigates and intensifies the pulmonary inflammatory cascade (Dechert et al., 2012; van der Heijden et al., 2009). Among these inflammatory factors, tumor necrosis factor-a (TNF-a), interleukin (IL)-4 (IL-4), IL-6, IL-8 and IL-10 are involved in cellular events and play key roles in pulmonary injury
Address for correspondence: Guoxiong Xu, MD, PhD, Center Laboratory, Jinshan Hospital, Fudan University, 1508 Longhang Road, Shanghai 201508, P.R. China. Tel: +86-21-34189990. Fax: +86-2167226910. E-mail: [email protected] Jie Shen, MD, Center of Emergency & Intensive Care Unit, Jinshan Hospital, Fudan University, 1508 Longhang Road, Shanghai 201508, P.R. China. Tel: +86-18930819779. Fax: +86-21-67226910. E-mail: [email protected]
Received 19 October 2013 Revised 28 November 2013 Accepted 2 December 2013 Published online 31 January 2014
(Bhatia & Moochhala, 2004; Hodge et al., 2002; Huaux et al., 2003; Lucas et al., 2009; Ware, 2005). It is well documented that the pro-inflammatory and antiinflammatory cytokines are mediated by the p38 mitogenactivated protein kinase (MAPK) and nuclear factor-kappa B (NF-kB) pathways. There are three MAPKs: extracellular signal-regulated kinases (ERK), c-Jun NH2-terminal kinases (JNK) and p38 MAPK (Johnson & Lapadat, 2002; Pearson et al., 2001). Among them, p38 MAPK regulates many cytokines in ALI/ARDS caused by various reasons (Arcaroli et al., 2001; Tamura et al., 1998). The activation of p38 MAPK signaling leads to the activation of NF-kB, a transcription factor, by modulating the phosphorylation of p65 NF-kB (Kweon et al., 2006), which plays a central role in the onset of inflammation and increases the level of inflammatory mediators (Park & Christman, 2006). Indeed, the suppression of the MAPK and NF-kB pathways contributes to the attenuation of inflammatory responses in ALI (Chi et al., 2013). The angiopoietin-Tie2 system is involved in the regulation of inflammation, hyperpermeability, vasoreactive stimuli and apoptosis in the endothelium (van der Heijden et al., 2009). Angiopoietin-1 (Ang1) reduces inflammation and vascular hyperpermeability, whereas angiopoietin-2 (Ang2) has an opposite effect on inflammation (Sandhu et al., 2004; Thurston et al., 2000). Both Ang1 and Ang2 are ligands for the receptor tyrosine kinase Tie2 (Tsigkos et al., 2003). However, Ang2 is able to antagonize the elevated Ang1-activation (Hansen et al.,
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2010). Ang1 functionally binds to and stimulates monocytes via p38, which is associated with pro-inflammatory cytokine TNF-a (Seok et al., 2013). Our previous study demonstrated that adenovirus-delivered Ang1 (Ad/Ang1) has beneficial effects on Psg-induced lung injury though it reduces endothelial permeability and inflammation in vivo (Shen et al., 2013). However, whether Ang1 inhibits Ang2 and whether Ang1 depresses the influence of the p38 MAPK pathway on TNF-a-induced NF-kB activation in Psg-induced ALI have not been investigated. In this study, we examined the relationship between Ang1 and inflammatory responses and studied the suppressive effects of Ad/Ang1 on NF-kB activation and phosphorylation of p38 (p-p38) associated with inflammatory responses in Psg-induced ALI.
Materials and methods Animals Sixty male Sprague-Dawley (SD) rats were used for the study and the experiment designs were the same as in our previous study (Shen et al., 2013). We randomly divided the animals into six groups: control (Air), adenovirus control (Air + Ad), adenovirus-Ang1 (Air + Ad/Ang1), Psg gas alone, Psg plus adenovirus control (Psg + Ad) and Psg plus adenovirus-Ang1 (Psg + Ad/Ang1). At least three animals were used in each group. The left lung from each rat was collected for bronchoalveolar lavage fluid (BALF), RNA or protein extraction. This work was approved by the Ethic Committee of Jinshan Hospital for Animal Care, according to the Guidelines provided by Chinese Academy of Medical Sciences.
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an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer’s instructions. RNA extraction and quantitative real-time RT-PCR Total RNA was extracted and real-time RT-PCR was performed as described previously. The primer sequences were as follow: rat Ang2 50 -GTCAACACCTTTATCCATGA CACCA-30 (forward) and 50 -GCAAGTGGTGACCTGGAAG TGAG-30 (reverse), rat NF-kB 50 –GGAGTACGACAACATC TCGTTGG-30 (forward) and 50 -TGTAGAGGTGTCGTCCCA TCGT-30 (reverse), rat p38 50 -ACCACGACCCTGATGATG AGC-30 (forward) and 50 -GGTCAGGCTCTTCCATTCGTC T-30 (reverse), rat GAPDH 50 -CCTCAAGATTGTCAGCAA T-30 (forward) and 50 -CCATCCAGCAGTCTTCTGAGT-30 (reverse). All experiments were conducted in triplicate and repeated three times. Western blot The lung tissues were homogenized in RIPA lysis buffer and equal amount of total protein was subjected to SDSPAGE and transferred to PVDF membranes as described previously (Shen et al., 2013). Anti-Ang2, anti-p38, antiphospho-p38, anti-NF-kB p65 and anti-GAPDH were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The primary antibody was incubated overnight at 4 C. The secondary antibody (anti-rabbit or anti-mouse HRPconjugated IgG) was incubated for one hour at room temperature. The specific bands were visualized using enhanced chemiluminescence reagents (GE Healthcare, Biosciences, Pittsburgh, PA).
Exposure to Psg Psg gas was prepared as described previously (Shen et al., 2013). The animals were exposed to the gas at a final concentration of 8.33 g/m3 in an enclosed cabinet for 5 min, placed in separate cabinets with fresh air and then sacrificed after 36 hours of Psg exposure. Treatment with Ad/Ang1 Ang1-overexpressing rats were established by an intravenous injection of Ad/Ang1 at an optimized dose of 1 108 pfu/ml via tail vein an hour post-Psg exposure as described previously (Shen et al., 2013). The animals were sacrificed after 36 hours of administration. Blood and BALF sample collection and lung tissue preparation The blood was collected from abdominal aorta 36 hours postPsg inhalation, and the BALF was collected from the left lung. The serum and BALF were used to measure the concentrations of Ang2 and inflammatory cytokines. The right lung was perfused with saline and used for RT-PCR and Western Blot analyses. Enzyme-linked immunosorbent assay The concentrations of Ang2, TNF-a, IL-4, IL-6, IL-8, and IL-10 in the serum and BALF were measured using
Statistical analyses All statistical analyses were carried out as described previously (Shen et al., 2013). The differences were considered significant at values of p50.05.
Results Reduction of Psg-induced Ang2 expression by Ad/Ang1 Following our previous work (Shen et al., 2013), we successfully replicated the model of Psg-induced ALI in rats, as demonstrating the overexpression of Ad/Ang1 in the lung detected by fluorescent microscopy and Western blot (data now shown). Since ALI is associated with a decrease in Ang1 and an increase in Ang2, we examined whether the overexpression of Ad/Ang1 reverses Psg-induced upregulation of Ang2 in ALI. We found that the level of Ang2 in the serum and BALF was significantly elevated after Psg exposure detected by ELISA (Figure 1A and B). However, this Psg-induced increase in Ang2 was reduced after Ad/Ang1 treatment. Up-regulation of Ang2 expression in the lungs was also detected at both mRNA and protein levels as assessed by qRT-PCR (Figure 1C) and Western blot (Figure 1D and E), respectively. This Psg-induced upregulation of Ang2 in ALI was significantly deceased after administration of Ad/Ang1.
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Figure 1. Detection of Ang2 in air- and Psg-exposed rats. The concentration of Ang2 in the serum (A) and BALF (B) was measured by ELISA. The Ang2 mRNA expression was determined by qRT-PCR (C) and the Ang2 protein expression was determined by Western blot (D, E). Data represent mean ± SEM. Different letters denote significance (p50.05, n ¼ 3 experiments). Air, air-exposed control; Air + Ad, air-exposed animal with adenovirus alone; Air + Ad/Ang1, air-exposed animal with adenovirus-delivered Ang1; Psg, Psg-exposed animal; Psg + Ad, Psg-exposed animal with adenovirus alone; Psg + Ad/Ang1, Psg-exposed animal with adenovirus-delivered Ang1.
Secreted inflammatory cytokines in the serum and BALF Pro-inflammatory cytokines TNF-a, IL-6 and IL-8 are critical factors and their concentrations are known to be altered in the process of ALI. Subsequently, we examine whether the levels of secreted pro-inflammatory cytokines in the serum and BALF are altered after Psg exposure. We found that secreted TNF-a, IL-6 and IL-8 levels, detected by ELISA, were significantly increased in the serum (Figure 2A, C and E) and BALF (Figure 2B, D and F) of Psg-exposed rats as compared with air-exposed rats. Interestingly, the increase in the levels of TNF-a, IL-6, and IL-8 after Psg exposure was significantly reversed after the treatment of Ad/Ang1 (Figure 2). It has been shown that IL-4 and IL-10 are critical anti-inflammatory cytokines during ALI (Dinarello, 2000).
Here we found that secreted IL-4 and IL-10 were also increased in the serum (Figure 3A and C) and BALF (Figure 3B and D) of Psg-exposed rats as compared with air-exposed rats. Though Ad/Ang1 reversed pro-inflammatory cytokines in Psg-induced ALI, statistical analysis showed that IL-4, which was upregulated after Psg exposure, was not significantly affected by Ad/Ang1 in the serum and BALF (Figure 3A and B). However, IL-10, which was upregulated after Psg exposure, was partially, but not significantly, reversed after the treatment of Ad/Ang1 in the serum and BALF (Figure 3C and D). Effect of Ad/Ang1 on the expression of p38 mRNA and protein in Psg-exposed rats The expression of p38 mRNA was not altered in the lungs of Psg-induced ALI rats (Figure 4A). The administration
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Figure 2. Measurement of secreted TNF-a, IL-6 and IL-8 levels in the serum and BALF by ELISA. The secreted TNF-a levels were detected in the serum (A) and BALF (B). The secreted IL-6 levels were detected in the serum (C) and BALF (D). The secreted IL-8 concentration was detected in rat serum (E) and BALF (F). Data represent mean ± SEM. Different letters denote significance (p50.05, n ¼ 3 experiments). Air, air-exposed control; Air + Ad, air-exposed animal with adenovirus alone; Air + Ad/Ang1, air-exposed animal with adenovirus-delivered Ang1; Psg, Psg-exposed animal; Psg + Ad, Psg-exposed animal with adenovirus alone; Psg + Ad/Ang1, Psg-exposed animal with adenovirus-delivered Ang1.
of Ad and Ad/Ang1 did not change the p38 mRNA expression (Figure 4A). By comparing total p38 protein expression between all the tested groups, we found that the expression of total p38 protein was quite stable. There was no significant difference in total p38 protein expression after Psg exposure with or without Ad/Ang1 treatment, compared with airexposed control (Figure 4B and C). However, we observed that phospho-p38 protein was significantly increased after Psg exposure. The increased phosphor-p38 in the Psg-exposed rats was partially, but significantly, reversed after Ad/Ang1 treatment (Figure 4B and D).
Effect of Ad/Ang1 on NF-kB mRNA and protein expression in Psg-exposed rats Next, we examined whether Ad/Ang1 affects the expression of p65 NF-kB. We found that NF-kB mRNA expression was significantly increased in Psg-expose rats compared with airexposed rats (Figure 5A). The administration of Ad/Ang1 significantly reduced Psg-induced NF-kB mRNA expression. We also observed that NF-kB protein expression increased after Psg exposure (Figure 5B and C). The increase in NF-kB protein expression in the Psg-exposed rats revised after
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Figure 3. Measurement of secreted IL-4 and IL-10 levels in the serum and BALF by ELISA. The secreted IL-4 levels were detected in the serum (A) and BALF (B). The secreted IL-10 levels were detected in the serum (C) and BALF (D). Data represent mean ± SEM. Different letters denote significance (p50.05, n ¼ 3 experiments). Air, air-exposed control; Air + Ad, air-exposed animal with adenovirus alone; Air + Ad/Ang1, air-exposed animal with adenovirus-delivered Ang1; Psg, Psg-exposed animal; Psg + Ad, Psg-exposed animal with adenovirus alone; Psg + Ad/Ang1, Psg-exposed animal with adenovirus-delivered Ang1.
Ad/Ang1 treatment (Figure 5B and C). Ad alone did not affect the expression of NF-kB.
Discussion In our previous study using Psg-induced ALI model, we demonstrated that Ad/Ang1 has a protective benefit which prevents the damage of the lung by stabilizing vascular endothelial growth factor (VEGF), suppressing leukocytes infiltration and reducing inflammation (Shen et al., 2013). In the present study, we successfully replicated the model of Psg-induced ALI in rats and examined the inflammatory cytokines in the serum and BALF with or without Ad/Ang1 treatment. The administration of Ad/Ang1 resulted in partial reversion of the adverse effects of Psg in ALI, especially for the inflammatory cytokines. We have also explored the effect of Ad/Ang1 on the regulation of NF-kB and the activation of p38 MAPK, both of which play important roles in regulating the inflammatory cytokines. Currently, there is no specific and effective treatment available for ALI. Adenoviral gene delivery as gene therapy has been used in several animal models (Jenkins et al., 2003; Suzuki et al., 2001). Previous reports from us and others showed that Ad/Ang1 inhibits the progression of the damage in ALI after Psg inhalation (Shen et al., 2013) and lipopolysaccharide instillation (Xu et al., 2011). It has been shown that
Ang1 and Ang2 are competitive binding to the tyrosine kinase receptor Tie2 (Tsigkos et al., 2003). Ang2 blocks the effects of Ang1 and antagonizes Tie2 (Hansen et al., 2010). Our previous and current studies show that Psg is an important modulator of the angiopoietin family by inhibiting Ang1 and Tie2 and stimulating the expression of Ang2. These results support the fact that angiopoietin/Tie2 system is important in mediating the pulmonary vascular and inflammatory responses. Current data confirmed the previous report (Sciuto et al., 2003) that TNF-a, IL-6 and IL-8 in the serum and BALF were significantly higher in Psg-exposed mice than airexposed controls. Time-course study showed that IL-10 was significantly higher in Psg-exposed, whereas IL-4 was significantly lower in the Psg-exposed mice than in the airexposed mice from 4 to 8 hours post-exposure (Sciuto et al., 2003). In our rat model, however, we found that both IL-4 and IL-10 were significantly increased at 36 hours post-Psg exposure. The treatment of Ad/Ang1 significantly decreased serum and BALF levels of TNF-a, Il-6 and IL-8, respectively, not IL-4 and IL-10, leading the speculation that Ad/Ang1 decreases pro-inflammatory cytokines, not anti-inflammatory cytokines, to reduce lung permeability and inflammation responses. The future study is needed to assess the temporal changes of pro- and anti-inflammatory markers in Psginduced ALI animals after Ad/Ang1 treatment.
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Figure 4. Expression of p38 mRNA and protein in the lung. (A) mRNA expression of p38 detected by qPCR. (B) Western blot detection for p38 protein. (C) Semi-quantification of total p38 protein in Western blot. (D) Semi-quantification of phosphorylated p38 protein in Western blot. Data represent mean ± SEM. Different letters denote significance (p50.05, n ¼ 3 experiments). Air, air-exposed control; Air + Ad, air-exposed animal with adenovirus alone; Air + Ad/Ang1, air-exposed animal with adenovirus-delivered Ang1; Psg, Psg-exposed animal; Psg + Ad, Psg-exposed animal with adenovirus alone; Psg + Ad/Ang1, Psg-exposed animal with adenovirus-delivered Ang1.
Previous studies found that the inhibition of the NF-kB and p38 MAPK pathways reduces TNF-a-stimulated IL-8 expression (Everhart et al., 2006; Han et al., 2009; Kuldo et al., 2005; Su et al., 2008). Ang1, as opposed to Ang2, promotes neutrophil IL-8 synthesis and release through the activation of the p42/44 MAPK pathway (Neagoe et al., 2012). NF-kB is a family of DNA-binding proteins that is required for the transcription of many pro-inflammatory mediators and plays an important role in the pathogenesis of ALI (Kim et al., 2006; Liu et al., 2008). The activation of NF-kB leads to coordinated expression of various mediators of inflammation and perpetuation of the inflammatory response (Oeckinghaus & Ghosh, 2009; Park & Christman, 2006). It has been shown that NF-kB activation correlates with severity of lung injury. Suppression in the NF-kB pathway is beneficial even after the onset of lung inflammation (Everhart et al., 2006) and, therefore, NF-kB is an obvious target for anti-inflammatory treatment (Aghai et al., 2006). Thus, downregulation of NF-kB activity is a useful strategy to regulate inflammatory response. Our studies indicated that the expression of NF-kB factor p65 increased in Psg-induced ALI. The increase in NF-kB may lead to an increase in pro-inflammatory cytokines. Administration of Ad/Ang1 decreases Psg-induced
NF-kB activation, which in turn, inhibits the production of TNF-a, IL-6 and IL-8. This result suggests that the suppression of the NF-kB signaling pathway to reduce the production of inflammatory mediators is one of the mechanisms of Ad/Ang1 therapy. Previous studies showed that the activation of p38 MAPK is important in inflammatory mediator induction and involved in the ALI process (Kaminska et al., 2009). For example, the p38 MAPK pathway is critically important in regulating IL-8 expression in HUVECs (Kuldo et al., 2005) and enhances the production of IL-4 and IL-10 (Yeh et al., 2007). Current study showed that Psg activated the p38 MAPK pathway by the increase of p38 phosphorylation rather than by the regulation of total p38 in the lungs. Administration of Ad/Ang1 markedly inhibited Psg-induced phosphorylation of p38. These results indicate that the effects of Ang1 on the production of pro-inflammatory mediators may occur by blocking the p38 MAPK signaling pathway. In conclusion, we have demonstrated that Ang1 inhibits pro-inflammatory mediators, such as TNF-a, IL-6 and IL-8, and has no effect on anti-inflammatory mediators, such as IL-4 and IL-10, in the serum and BALF of ALI rats. This inhibitory action of Ang1 was mediated by the suppression of
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the NF-kB and p38 MAPK pathways, leading to the attenuation of inflammatory responses. Thus, Ad/Ang1 may provide a useful tool for the effective treatment in Psginduced ALI.
Declaration of interest The authors report no declarations of interest. This research was supported by National Youth Nature Science Foundation of China (No. 81101412) to DKH and a start-up fund of research from Jinshan Hospital (2012-2) to GX.
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Figure 5. Expression of NF-kB mRNA and protein in the lung. (A) mRNA expression of NF-kB by qPCR. (B) Western blot detection for NF-kB protein. (C) Semi-quantification of NF-kB protein in Western blot. Data represent mean ± SEM. Different letters denote significance (p50.05, n ¼ 3 experiments). Air, air-exposed control; Air + Ad, airexposed animal with adenovirus alone; Air + Ad/Ang1, air-exposed animal with adenovirus-delivered Ang1; Psg, Psg-exposed animal; Psg + Ad, Psg-exposed animal with adenovirus alone; Psg + Ad/Ang1, Psg-exposed animal with adenovirus-delivered Ang1.
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