Respiratory Physiology & Neurobiology 201 (2014) 7–14
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Keratinocyte growth factor-2 is protective in lipopolysaccharide-induced acute lung injury in rats Lin Tong a,1 , Jing Bi a,1 , Xiaodan Zhu a , Guifang Wang a , Jie Liu a , Linyi Rong a , Qin Wang a , Nuo Xu a , Ming Zhong b , Duming Zhu b , Yuanlin Song a,c,d,∗∗ , Chunxue Bai a,∗ a
Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, PR China Division of Critical Care Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, PR China c Shanghai Public Health Clinical Center, Shanghai 201508, PR China d Zhongshan Hospital, Qingpu Branch, Fudan University, Shanghai 201700, PR China b
a r t i c l e
i n f o
Article history: Accepted 20 June 2014 Available online 26 June 2014 Keywords: Acute lung injury Keratinocyte growth factor-2 Lipopolysaccharide Epithelial cells Pulmonary surfactant proteins
a b s t r a c t Keratinocyte growth factor-2 (KGF-2) plays a key role in lung development, but its role in acute lung injury has not been well characterized. Lipopolysaccharide instillation caused acute lung injury, which significantly elevated lung wet-to-dry weight ratio, protein and neutrophils in bronchoalveolar lavage fluid (BALF), inhibited surfactant protein A and C expression in lung tissue, and increased pathological injury. Pretreatment with KGF-2 improved the above lung injury parameters, partially restored surfactant protein A and C expression, and KGF-2 given 2–3 days before LPS challenge showed maximum lung injury improvement. Pretreatment with KGF-2 also markedly reduced the levels of TNF-␣, MIP-2, IL-1 and IL-6 in BALF and the levels of IL-1 and IL-6 in lung tissue. Histological analysis showed there was increased proliferation of alveolar type II epithelial cells in lung parenchyma, which reached maximal 2 days after KGF-2 instillation. Intratracheal administration of KGF-2 attenuates lung injury induced by LPS, suggesting KGF-2 may be potent in the intervention of acute lung injury. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are common and potentially lethal respiratory diseases (Wheeler and Bernard, 2007). By new definition, ALI is the mild form of ARDS, which is also characterized by heterogeneous alveolar epithelium and endothelium damage with accumulation of plasma proteins and flooding in the alveoli causing refractory hypoxemic respiratory failure. Keratinocyte growth factor-2 (KGF-2), namely fibroblast growth factor-10, has been shown to mediate epithelial–mesenchymal interactions, which are essential in lung development (Benjamin et al., 2007; Hyatt et al., 2004; Ware and Matthay, 2002). Recently KGF-2 showed preventive effects on lung injury from various
∗ Corresponding author at: 180 Fenglin Road, Shanghai 200032, PR China. Tel.: +86 21 64041990x3077; fax: +86 21 54961729. ∗∗ Corresponding author at: 180 Fenglin Road, Shanghai 20003, PR China. Tel.: +86 21 64041990x2422; fax: +86 21 54961729. E-mail addresses: [email protected] (Y. Song), [email protected] (C. Bai). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.resp.2014.06.011 1569-9048/© 2014 Elsevier B.V. All rights reserved.
stresses (Kim et al., 2009; Gupte et al., 2009; Upadhyay et al., 2004, 2005). With similarities to keratinocyte growth factor (KGF), KGF-2 is a heparin-binding protein predominantly expressed by mesenchymal cells. It binds with high affinity to a spliced variant of fibroblast growth factor receptor 2-IIIb which is expressed solely on epithelial cells. KGF-2 has a weaker affinity for fibroblast growth factor receptor 1-IIIb, which is expressed on epithelial/endothelial cells and is a functional transmembrane receptor for KGF-2 but not for KGF (Ware and Matthay, 2002; Beer et al., 2000; Igarashi et al., 1998; Yamasaki et al., 1996; Emoto et al., 1997). Thus, KGF acts specifically on epithelial cells, whereas KGF-2 seems to have broader cell type specificity (Kim et al., 2009). The role of KGF in acute lung injury has been extensively studied since 1990s. But the concern about the carcinogenetic effect of KGF hindered its further translation to bedside (Chang et al., 2009; Mehta et al., 2010; Lin et al., 2011; Zang et al., 2009). However, KGF-2 had no in vitro or in vivo proliferative effects on human epithelial-like tumors (Alderson et al., 2002). This is critical to the safety profile of KGF-2 and provides its potential therapeutic feasibility. The above studies suggest that KGF-2 might play an important role in ALI, but there are few studies directly assessed this possibility of exogenous KGF-2 in vivo. In this study, we used the ALI model
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induced by intratracheal instillation of lipopolysaccharide (LPS) to evaluate the effect of exogenous KGF-2. 2. Methods
was graded according to a five-point scale: 1, no injury; 2, injury to 25% of the field; 3, injury to 50% of the field; 4, injury to 75% of the field; 5, diffuse injury (Su et al., 2004b). All samples were examined by 3 pathologists blinded to the experimental procedures and the mean score was used for comparison.
2.1. Animals Seventy male specific pathogen free Sprague-Dawley rats weighing 200–250 g were used in the experiments. Animals were maintained in the animal facility at Fudan University with clean, controlled temperature and independent ventilation environment. The animals had free access to food and water. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Zhongshan Hospital, Fudan University. All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, and efforts were made to minimize suffering and pain of the animals, and number in each group in our study.
2.6. Immunohistochemistry analyses
2.2. Animal treatment
Total RNA was extracted using the Trizol reagent (Invitrogen, CA, USA). RNAs were reverse-transcribed, and real-time polymerase chain reaction was performed using primers for the examined transcripts (Table 1).
Rats were anesthetized with intraperitoneal injection of chloral hydrate (300 mg/kg). The instillate (recombinant human KGF-2 (rhKGF-2, recombinant product from E. coli after purification, MW 19.3 kDa, manufactured by Newsummit Pharmaceutical Company, Shanghai, China), phosphate buffered saline (PBS), or LPS) was then injected into the trachea using an 18G catheter attached to a 1-ml syringe as previously described (Su et al., 2004a). RhKGF-2 at a dose of 5 mg/kg was instilled through the catheter into the lungs of rats. The dose of KGF-2 used in the present study was based on previous experiments (She et al., 2012; Fang et al., 2014). Control animals received equal volume of PBS. After certain days (1, 2, 3, or 5 days), rats were intratracheally instilled with either 5 mg/kg LPS (E. coli O55:B5; Sigma, St. Louis, MO) dissolved in 0.3 ml PBS or vehicle (PBS). The rats were euthanized 24 h after LPS instillation with an intraperitoneal injection of urethane (1.5 g/kg) and exsanguinated through the cervical artery after sampling for arterial blood gas analysis. After wet weights were measured, the middle lobe of the right lung were placed in an oven at 60 ◦ C for 72 h to allow determination of the wet-to-dry weight ratio.
Immunostaining of lung tissue was done using 5-m paraffin sections. Labeling was obtained by incubation with rabbit anti-prosurfactant protein C (proSP-C) polyclonal antibody (Millipore, MA, USA), or mouse anti-Ki67 monoclonal antibody (Dako, Denmark). Sections were covered with DAB tetrahydroxychloride, and counterstained with hematoxylin.
2.7. Quantitative real-time polymerase chain reaction analyses
2.8. Western blot analyses Lung tissues were homogenized and 20 g of protein was electrophoresed. Membranes were exposed overnight at 4 ◦ C to rabbit anti-proSP-C polyclonal antibody (Millipore, MA, USA), or rabbit anti-surfactant protein A (SP-A) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, USA), or rabbit anti--actin polyclonal antibody (Abcam, UK) as loading control.
2.9. Statistical analyses Each point corresponds to the mean ± SEM. Statistical differences were determined using the one-way analysis of variance, and p < 0.05 was considered significant. Individual groups were compared using the unpaired Student’s t test.
2.3. Total cell count and differential cell count The bronchoalveolar lavage was done in the left lung. Bronchoalveolar lavage fluid (BALF) samples were centrifuged, and the pellet was resuspended in PBS. The total number of nucleated cells in BALF was counted with a hemocytometer. Then the resuspended BALF was centrifuged onto slides and stained with Wright–Giemsa stain. The slides were used to quantify neutrophil number by counting a total of 200 cells per slide. 2.4. Protein concentration and cytokine levels in BALF BALF protein concentration was measured using bicinchoninic acid protein assay (Thermo Fisher Scientific, MA, USA). Tumor necrosis factor-␣ (TNF-␣), interleukin-1 (IL-1), interleukin-6 (IL6), and macrophage inflammatory protein-2 (MIP-2) levels in BALF were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA) according to manufacture’s guideline. 2.5. Lung morphometry analyses The lungs were fixed in 10% formalin, and 5 m sections were cut for hematoxylin and eosin staining. Lung injury was scored according to the following variables: alveolar and interstitial edema, neutrophil infiltration and hemorrhage. Each variable
3. Results 3.1. KGF-2 time-dependent protective effects on LPS-induced ALI Intratracheal instillation of LPS induced ALI which is represented by a significant decrease in arterial PO2 , and a significant increase in BALF protein concentration, BALF total cell number, BALF neutrophil number, lung wet-to-dry weight ratio, and lung injury score 24 h after LPS administration compared to that in rats receiving PBS as control (Fig. 1). In order to define the best response timing for rhKGF-2 on lung injury prevention, rhKGF-2 was instilled at different time points. Indeed, intratracheal instillation of rhKGF-2 1–5 days before LPS challenge showed different improvements in lung injury compared to animals without pretreatment (Fig. 1). Pretreatment with rhKGF2 two or three days before LPS challenge resulted in a significant improvement in lung injury parameters compared with that in control rats receiving no pretreatment. Although there is no significant difference of above parameters between 2 and 3 days before LPS instillation, absolute number indicates pretreatment 3 days prior to insult might be slightly more effective than the other time points. So we decided to further investigate the protective effect of rhKGF-2 pretreatment 3 days before challenge in LPS-induced ALI.
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Table 1 Primer sequences for quantitative real-time polymerase chain reaction. Genes
Primers (5 –3 )
GenBank accession no.
Amplicon size (bp)
Interleukin-1
F-GCTGTGGCAGCTACCTATGTCTTGR-AGGTCGTCATCATCCCACGAGF-CCACTTCACAAGTCGGAGGCTTAR-GTGCATCATCGCTGTTCATACAATCF-AGGAGCTCCAGACTGAACTCTATGAR-CTGACTGCCCATTGGTGGAAF-GGGCCTTCACATGAGTCAGAAACR-CGATGCCAGTGGAGCCAATAF-GGAGATTACTGCCCTGGCTCCTAR-GACTCATCGTACTCCTGCTTGCTG-
NM 031512
120
NM 012589.1
108
NM 017329
123
NM 017342
140
NM 031144.2
150
Interleukin-6 Surfactant protein A Surfactant protein C -Actin
3.2. Cytokines and pathological changes with KGF-2 pretreatment 3 days before LPS challenge RhKGF-2 was administrated three days before LPS challenge. Four groups of rats, with/without LPS and with/without rhKGF-2 were compared to each other for lung injury measurement. LPS instillation significantly increased the levels of TNF-␣, IL-1, IL-6, and MIP-2 in BALF, while pretreatment of rhKGF-2 significantly reduced the levels of these cytokines (Fig. 2). The mRNA expression of IL-1 and IL-6 in lung tissue was similarly reduced with rhKGF2 (Fig. 2). Representative histological features of LPS-induced ALI were protein-rich edema fluid in alveolar space, leukocytes accumulation, hemorrhage and bronchial wall thickening with
inflammatory cells. Those changes were improved by rhKGF-2 pretreatment, and resulted in a lower score in lung injury than the vehicle pretreated groups (Fig. 3). 3.3. The change of surfactant proteins It was shown that the mRNA expression levels of SP-A and surfactant protein C (SP-C) were significantly decreased with LPS instillation, while rhKGF-2 pretreatment three days before partially restored the expression of SP-A and SP-C (Fig. 4). The protein expression level was measured, and the similar pattern was shown as the mRNA level (Fig. 4). Notably, rhKGF-2 administration alone increased the expression of SP-A and SP-C.
Fig. 1. Effect of rhKGF-2 on lung injury indices following intratracheal LPS. RhKGF-2 was adminstrated 1 (1d), 2 (2d), 3 (3d), or 5 (5d) days before LPS challenge, and no pretreatment was given to the control group (challenged with PBS instead of LPS) and LPS group(receiving 5 mg/kg LPS only). The effects of rhKGF-2 pretreatment on arterial PO2 (PaO2 ) (A), BALF total cell count (B) and neutrophil count (C), BALF protein concentration (D), lung wet-to-dry weight ratio (E), and lung injury score (F) following intratracheal LPS were evaluated. Data are expressed as mean ± SEM (n = 4 for each group). *p < 0.05, **p < 0.01 compared with control, and †p < 0.05, ††p < 0.01 compared with the LPS group.
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Fig. 2. The levels of cytokines in bronchoalveolar lavage fluid (BALF) and lung tissue. Pretreatment with rhKGF-2 markedly reduced the levels of tumor necrosis factor-␣ (TNF-␣) (A), macrophage inflammatory protein-2 (MIP-2) (B), interleukin (IL)-1 (C) and IL-6 (D) in BALF and the mRNA expression levels of IL-1 (E) and IL-6 (F) in lung tissue. Data are expressed as mean ± SEM (n = 6 for each group). *p < 0.05, **p < 0.01 compared with control, and †p < 0.05, ††p < 0.01 compared with the LPS group.
3.4. The proliferation of alveolar type II epithelial cells 2–6 days after KGF-2 treatment
hyperplastic alveolar epithelial cells contained one or more lamellar inclusions (Fig. 5).
Immunohistochemistry staining was conducted for proSP-C, which is expressed solely on alveolar type II epithelial cells, and Ki67, a proliferation marker of cells. RhKGF-2 was injected intratracheally at a single dose of 5 mg/kg, and the lungs at 2, 3, 4, 6 days after injection were harvested. Immunoreactive proSP-C was demonstrable in hyperplastic alveolar lining cells 2 days after rhKGF-2 treatment. The number of proSP-C positive cells in rhKGF2 treated lungs was one-fold increased at day 2 compared to vehicle treated lungs, then slightly decreased and normalized at day 6 (Fig. 5). Ki67 expression was prominently detected in alveolar type II epithelial cells at 2 day after the intratracheal injection of rhKGF-2, a time point at which epithelial cell hyperplasia was morphologically recognizable. And the Ki67 expression was not prominent at day 6. The type II pneumocytes in the lungs of control rats showed only rare expression of Ki67. Meanwhile, rhKGF-2 also caused an increase of Ki67 expression in bronchiolar airways that was more focal and less striking than that in the alveolar type II epithelial cells (Fig. 5). The lungs of rhKGF-2 treated rats were ultrastructurally examined 2 days after intratracheal injection, and it showed that the
4. Discussion We have shown that intratracheal administration of rhKGF2 at a dose of 5 mg/kg body weight two or three days prior to insult had significant protective effects on LPS-induced ALI in rats. Additional studies showed that pretreatment of rhKGF-2 partially restored lung SP-A and SP-C expression after LPS challenge. Our study showed that there was proliferation of alveolar type II epithelial cells in lung parenchyma after rhKGF-2 instillation. These data demonstrate that KGF-2 pretreatment can ameliorate the LPSinduced ALI effectively. A series of studies have demonstrated that KGF has potential protective effects in ALI induced by various stresses (FrancoMontoya et al., 2009; Ulrich et al., 2005; Terry et al., 2004; Welsh et al., 2000; Viget et al., 2000; Yano et al., 1996; Sugahara et al., 1998). Due to its huge similarities to KGF (Igarashi et al., 1998), KGF2 was supposed to be even more potent in protection of lung injury because unlike KGF, KGF-2 receptor was expressed on both epithelial and endothelial cells (Huang and Berkland, 2009; She et al., 2012). Gupte et al. (2009) recently reported the anti-fibrotic effect of KGF-2 on bleomycin-induced pulmonary fibrosis. Our group also
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Fig. 3. Histological quantitation of lung injury following LPS challenge. Sections of hematoxylin and eosin-stained lung tissue were examined under light microscopy (5×), and damage was recorded using the parameters described in Section 2. (A) Section from control animals receiving intratracheal PBS showing alveolar spaces free of inflammatory cell infiltration, with no evidence of hemorrhage or edema. (B) Pretreatment of rhKGF-2 without LPS challenge showed similar appearance as the lung of (A). (C) LPS administration (5 mg/kg) results in the appearance of edematous areas, with a large number of inflammatory cells infiltration. (D) Pretreatment of rhKGF-2 ameliorates lung damage following LPS instillation. Sections are representative of 6 animals examined per group.
showed significant improvement in survival and lung injury in a high altitude pulmonary edema rat model with protection on epithelium and endothelium stress (She et al., 2012). The mechanisms underlying the protective effect of KGF-2 in ALI especially LPS-induced ALI remain incompletely understood
but are probably multiple. The present study investigated the role of KGF-2 on lung inflammation and permeability caused by LPS instillation, the mitogenic effect on alveolar type II epithelial cells and changes in the production of surfactant proteins by epithelial cells.
Fig. 4. The mRNA and protein expression levels of surfactant protein A (SP-A) and surfactant protein C (SP-C) in lung tissue. (A) Relative mRNA expression level of SP-A in lung tissue. (B) Relative mRNA expression level of SP-C in lung tissue. (C) Western blot analysis of SP-A expression level in lung tissue. (D) Western blot analysis of prosurfactant protein C (proSP-C) expression level in lung tissue. Data are expressed as mean ± SEM (n = 6 for each group). **p < 0.01 compared with control, ††p < 0.01 compared with the LPS group.
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Fig. 5. KGF-2 stimulates alveolar type II epithelial cell proliferation. Labeling was obtained by incubation with anti-prosurfactant protein C (proSP-C) antibody (A) or anti-Ki67 antibody (B–D). Sections were covered with DAB tetrahydroxychloride, and counterstained with hematoxylin. (A) Immunoreactive proSP-C is demonstrable in hyperplastic alveolar lining cells 2 days after intratracheal injection of 5 mg/kg rhKGF-2 (400×). (B) Ki67 expression was prominently detected in alveolar type II epithelial cells 2 days after the intratracheal injection of rhKGF-2 (400×). (C) The alveolar type II epithelial cells in the lungs of control rats do not demonstrate any Ki67 expression in many microscopic fields, although occasional alveolar epithelial cells did express Ki67 (400×). (D) RhKGF-2 causes an increase in Ki67 expression in bronchiolar airway epithelium 2 days after intratracheal injection of rhKGF-2 (200×). (E) An electron micrograph (6000×) shows a group of hyperplastic alveolar epithelial cells containing cytoplasmic lamellar inclusions 2 days after intratracheal injection of rhKGF-2. The lamellar structure of the inclusions is appreciated at higher magnification (20,000×).
4.1. KGF-2 attenuates the inflammatory reaction in LPS-induced ALI Intratracheal instillation of LPS induces a rapid influx of neutrophils into lungs. This study consistently demonstrated that LPS administration significantly increased the neutrophil number in
BALF, while KGF-2 pretreatment markedly reduced the increase of neutrophil number in BALF, suggesting that KGF-2 pretreatment attenuates LPS-induced lung inflammation at least partly through reduced recruitment of neutrophils. Some cytokines play key roles in the initiation and development of lung inflammation including TNF-␣, IL-1, IL-6, and MIP-2
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(Strieter et al., 1999). LPS has been proved to induce secretion of these cytokines in vivo and in vitro (Su et al., 2004b; Suzuki et al., 2000). In this study, TNF-␣, IL-1, IL-6 and MIP-2 levels were markedly decreased in rats with KGF-2 pretreatment. Thus it suggests that KGF-2 pretreatment may suppress LPS-induced lung inflammation through inhibited production of cytokines and the consequent reduction of neutrophil infiltration. 4.2. KGF-2 reduces lung epithelial permeability after LPS challenge Intratracheal instillation of LPS has been shown to increase pulmonary endothelial and epithelial permeability, resulting in pulmonary edema (Su et al., 2004b). We recently found that KGF-2 pretreatment significantly reduced the hypoxia-induced increase in endothelial permeability both in vitro and in vivo via downregulation of apoptosis (She et al., 2012), while in this study, KGF-2 pretreatment attenuated the LPS-induced increases in BALF total protein level and lung wet-to-dry weight ratio, suggesting the protective effects of KGF-2 on lung epithelial barrier. 4.3. KGF-2 stimulates alveolar type II epithelial cell proliferation It has been reported that KGF is a growth factor for alveolar type II epithelial cells in vivo and in vitro (Ulich et al., 1994; Morikawa et al., 2000), and the maximum paracrine mitogenic effects of KGF on epithelium was 2–3 days after pretreatment (Ulich et al., 1994). However, the role of exogenous KGF-2 in alveolar epithelial proliferation has not been reported yet. Our data suggests that there is a substantial increased number of alveolar type II epithelial cells in lung parenchyma 2 days after KGF-2 treatment, and the extent of alveolar type II epithelial cell hyperplasia was similar to that triggered by KGF. The histologic manifestation of the alveolar epithelium on day 6 is much similar to the appearance of normal lungs, suggesting temporal changes of alveolar type II epithelial cells after KGF-2 pretreatment and its potential safety in future clinical implications. 4.4. KGF-2 restores SP- A and SP-C synthesis after LPS stimulation Surfactant is a complex mixture of lipid and protein, secreted by alveolar type II epithelial cells, and regulates surface tension at the pulmonary air–liquid interface to prevent alveolar collapse (Whitsett and Weaver, 2002). Thus we examined the expression of surfactant proteins in lung tissue and demonstrated that KGF-2 pretreatment partially restored SP-A and SP-C expression. However, there was no remarkable change of SP-B before and after KGF-2 treatment (data not shown). The increased expression of surfactant proteins might be due to alveolar type II epithelial cell proliferation and differentiation, and the change of intracellular surfactant proteins per single cell needs to be further addressed. This increase in surfactant proteins production might indirectly prevent a permeability defect, and might be a potential mechanism for KGF-2. Lung epithelial and endothelial repair play important roles in recovery of ALI. Recent studies have shown that stem cells promote lung injury recover through parasecretion of KGF (Lee et al., 2009). Both exogenous KGF and KGF gene delivery via mesenchymal stem cells can protect against LPS-induced ALI (Chen et al., 2013; Hu et al., 2010). The clinical application of KGF has been brought to frontier, since KGF has presently planned clinical randomized controlled trials in ALI to reduce pulmonary dysfunction (Cross et al., 2013). Our studies support the potential use of KGF-2 in prophylactic treatment of ALI. Intratracheal administration of KGF-2 in clinical scenario might be a good safe strategy, because topical application can reduce the side effects of systemic use. KGF-2 given 2–3 days before LPS challenge showed maximum lung injury improvement.
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This phenomenon suggests minimum of 2 days is need to allow KGF-2 reaching full effects and the duration of KGF-2 is also short which can be used in disease treatment without too much concern of the safety due to quick metabolism. Although in this study the data support the prophylactic use of KGF-2 in ALI induced by infectious insults, it should be noted that the animal model of ALI is of much limitation. This ALI model is an acute model, the lung injury recovers or deteriorates within several days. Thus when applying KGF-2 after lung injury, the animals recover or deteriorate before KGF-2 could reach its best effect. This might be the key reason why KGF-2 application after injury could not show marked improvement in lung injury. But this is quite different with bedside situation, because patients of ALI/ARDS experience much longer duration of disease. There might be enough time for KGF-2 to exert its multiple functions on modifying lung homeostasis to promote the recovery from ALI. However, due to the differences between humans and animals, the effects and mechanisms of KGF-2 in alveolar epithelium-capillary endothelium barrier repair after lung injury still needs further investigation. The possibility that KGF-2 may initiate excessive fibrosis after ALI should be a concern when considering the clinical use of KGF2. She et al. (2012) observed the long-term (1 and 2 months) effects of KGF-2 on multiple organs including lung, brain, liver, heart and kidney, and demonstrated that the tissues of those organs were normal and without excessive fibrosis. Furthermore, previous study showed that overexpression of KGF-2 during both inflammatory and fibrotic phases attenuated bleomycin-induced pulmonary fibrosis in mice (Gupte et al., 2009). Therefore, it seems that KGF-2 is a safe agent for treating patients with ALI/ARDS. However, it still needs to be more thoroughly studied as to the long-term effects in clinical trials for safety data in patients and to combinations with other agents used in ALI/ARDS, such as surfactant instillations and steroids. In conclusion, our study demonstrates the protective effect of KGF-2 on LPS-induced ALI, which may be mediated by reduction of pro-inflammatory cytokine levels and inflammatory cell infiltration, restored synthesis of SP-A and SP-C, and proliferation of alveolar type II epithelial cells. Therefore, KGF-2 may have potential clinical application as a prophylactic treatment of ALI. Acknowledgments We acknowledge Shanghai Newsummit Biopharma Co. for generously supplying rhKGF-2, Zude Xu, Shaohua Lu and Haiying Zeng (Fudan University) for the assistance in histological analyses. This research was supported by Shanghai Leading Academic Discipline Project (B115), and National Natural Science Foundation of China (30930090, 81100046, 81170056). Dr. Yuanlin Song was supported by the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning and Key Medical grant from Shanghai Science and Technology Committee (11411951102, 12JC1402300). References Alderson, R., Gohari-Fritsch, S., Olsen, H., Roschke, V., Vance, C., Connolly, K., 2002. In vitro and in vivo effects of repifermin (keratinocyte growth factor-2 KGF-2) on human carcinoma cells. Cancer Chemother. Pharmacol. 50, 202–212. Beer, H.D., Vindevoghel, L., Gait, M.J., Revest, J.M., Duan, D.R., Mason, I., Dickson, C., Werner, S., 2000. Fibroblast growth factor (FGF) receptor 1-IIIb is a naturally occurring functional receptor for FGFs that is preferentially expressed in the skin and the brain. J. Biol. Chem. 275, 16091–16097. Benjamin, J.T., Smith, R.J., Halloran, B.A., Day, T.J., Kelly, D.R., Prince, L.S., 2007. FGF10 is decreased in bronchopulmonary dysplasia and suppressed by Toll-like receptor activation. Am. J. Physiol. Lung Cell Mol. Physiol. 292, L550–L558. Chang, H.L., Sugimoto, Y., Liu, S., Wang, L.S., Huang, Y.W., Ye, W., Lin, Y.C., 2009. Keratinocyte growth factor (KGF) regulates estrogen receptor-alpha (ER-alpha) expression and cell apoptosis via phosphatidylinositol 3-kinase (PI3K)/Akt pathway in human breast cancer cells. Anticancer Res. 29, 3195–3205.
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Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Keratinocyte growth factor-2 is protective in lipopolysaccharide-induced acute lung injury in rats Lin Tong a,1 , Jing Bi a,1 , Xiaodan Zhu a , Guifang Wang a , Jie Liu a , Linyi Rong a , Qin Wang a , Nuo Xu a , Ming Zhong b , Duming Zhu b , Yuanlin Song a,c,d,∗∗ , Chunxue Bai a,∗ a
Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, PR China Division of Critical Care Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, PR China c Shanghai Public Health Clinical Center, Shanghai 201508, PR China d Zhongshan Hospital, Qingpu Branch, Fudan University, Shanghai 201700, PR China b
a r t i c l e
i n f o
Article history: Accepted 20 June 2014 Available online 26 June 2014 Keywords: Acute lung injury Keratinocyte growth factor-2 Lipopolysaccharide Epithelial cells Pulmonary surfactant proteins
a b s t r a c t Keratinocyte growth factor-2 (KGF-2) plays a key role in lung development, but its role in acute lung injury has not been well characterized. Lipopolysaccharide instillation caused acute lung injury, which significantly elevated lung wet-to-dry weight ratio, protein and neutrophils in bronchoalveolar lavage fluid (BALF), inhibited surfactant protein A and C expression in lung tissue, and increased pathological injury. Pretreatment with KGF-2 improved the above lung injury parameters, partially restored surfactant protein A and C expression, and KGF-2 given 2–3 days before LPS challenge showed maximum lung injury improvement. Pretreatment with KGF-2 also markedly reduced the levels of TNF-␣, MIP-2, IL-1 and IL-6 in BALF and the levels of IL-1 and IL-6 in lung tissue. Histological analysis showed there was increased proliferation of alveolar type II epithelial cells in lung parenchyma, which reached maximal 2 days after KGF-2 instillation. Intratracheal administration of KGF-2 attenuates lung injury induced by LPS, suggesting KGF-2 may be potent in the intervention of acute lung injury. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are common and potentially lethal respiratory diseases (Wheeler and Bernard, 2007). By new definition, ALI is the mild form of ARDS, which is also characterized by heterogeneous alveolar epithelium and endothelium damage with accumulation of plasma proteins and flooding in the alveoli causing refractory hypoxemic respiratory failure. Keratinocyte growth factor-2 (KGF-2), namely fibroblast growth factor-10, has been shown to mediate epithelial–mesenchymal interactions, which are essential in lung development (Benjamin et al., 2007; Hyatt et al., 2004; Ware and Matthay, 2002). Recently KGF-2 showed preventive effects on lung injury from various
∗ Corresponding author at: 180 Fenglin Road, Shanghai 200032, PR China. Tel.: +86 21 64041990x3077; fax: +86 21 54961729. ∗∗ Corresponding author at: 180 Fenglin Road, Shanghai 20003, PR China. Tel.: +86 21 64041990x2422; fax: +86 21 54961729. E-mail addresses: [email protected] (Y. Song), [email protected] (C. Bai). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.resp.2014.06.011 1569-9048/© 2014 Elsevier B.V. All rights reserved.
stresses (Kim et al., 2009; Gupte et al., 2009; Upadhyay et al., 2004, 2005). With similarities to keratinocyte growth factor (KGF), KGF-2 is a heparin-binding protein predominantly expressed by mesenchymal cells. It binds with high affinity to a spliced variant of fibroblast growth factor receptor 2-IIIb which is expressed solely on epithelial cells. KGF-2 has a weaker affinity for fibroblast growth factor receptor 1-IIIb, which is expressed on epithelial/endothelial cells and is a functional transmembrane receptor for KGF-2 but not for KGF (Ware and Matthay, 2002; Beer et al., 2000; Igarashi et al., 1998; Yamasaki et al., 1996; Emoto et al., 1997). Thus, KGF acts specifically on epithelial cells, whereas KGF-2 seems to have broader cell type specificity (Kim et al., 2009). The role of KGF in acute lung injury has been extensively studied since 1990s. But the concern about the carcinogenetic effect of KGF hindered its further translation to bedside (Chang et al., 2009; Mehta et al., 2010; Lin et al., 2011; Zang et al., 2009). However, KGF-2 had no in vitro or in vivo proliferative effects on human epithelial-like tumors (Alderson et al., 2002). This is critical to the safety profile of KGF-2 and provides its potential therapeutic feasibility. The above studies suggest that KGF-2 might play an important role in ALI, but there are few studies directly assessed this possibility of exogenous KGF-2 in vivo. In this study, we used the ALI model
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induced by intratracheal instillation of lipopolysaccharide (LPS) to evaluate the effect of exogenous KGF-2. 2. Methods
was graded according to a five-point scale: 1, no injury; 2, injury to 25% of the field; 3, injury to 50% of the field; 4, injury to 75% of the field; 5, diffuse injury (Su et al., 2004b). All samples were examined by 3 pathologists blinded to the experimental procedures and the mean score was used for comparison.
2.1. Animals Seventy male specific pathogen free Sprague-Dawley rats weighing 200–250 g were used in the experiments. Animals were maintained in the animal facility at Fudan University with clean, controlled temperature and independent ventilation environment. The animals had free access to food and water. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Zhongshan Hospital, Fudan University. All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, and efforts were made to minimize suffering and pain of the animals, and number in each group in our study.
2.6. Immunohistochemistry analyses
2.2. Animal treatment
Total RNA was extracted using the Trizol reagent (Invitrogen, CA, USA). RNAs were reverse-transcribed, and real-time polymerase chain reaction was performed using primers for the examined transcripts (Table 1).
Rats were anesthetized with intraperitoneal injection of chloral hydrate (300 mg/kg). The instillate (recombinant human KGF-2 (rhKGF-2, recombinant product from E. coli after purification, MW 19.3 kDa, manufactured by Newsummit Pharmaceutical Company, Shanghai, China), phosphate buffered saline (PBS), or LPS) was then injected into the trachea using an 18G catheter attached to a 1-ml syringe as previously described (Su et al., 2004a). RhKGF-2 at a dose of 5 mg/kg was instilled through the catheter into the lungs of rats. The dose of KGF-2 used in the present study was based on previous experiments (She et al., 2012; Fang et al., 2014). Control animals received equal volume of PBS. After certain days (1, 2, 3, or 5 days), rats were intratracheally instilled with either 5 mg/kg LPS (E. coli O55:B5; Sigma, St. Louis, MO) dissolved in 0.3 ml PBS or vehicle (PBS). The rats were euthanized 24 h after LPS instillation with an intraperitoneal injection of urethane (1.5 g/kg) and exsanguinated through the cervical artery after sampling for arterial blood gas analysis. After wet weights were measured, the middle lobe of the right lung were placed in an oven at 60 ◦ C for 72 h to allow determination of the wet-to-dry weight ratio.
Immunostaining of lung tissue was done using 5-m paraffin sections. Labeling was obtained by incubation with rabbit anti-prosurfactant protein C (proSP-C) polyclonal antibody (Millipore, MA, USA), or mouse anti-Ki67 monoclonal antibody (Dako, Denmark). Sections were covered with DAB tetrahydroxychloride, and counterstained with hematoxylin.
2.7. Quantitative real-time polymerase chain reaction analyses
2.8. Western blot analyses Lung tissues were homogenized and 20 g of protein was electrophoresed. Membranes were exposed overnight at 4 ◦ C to rabbit anti-proSP-C polyclonal antibody (Millipore, MA, USA), or rabbit anti-surfactant protein A (SP-A) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, USA), or rabbit anti--actin polyclonal antibody (Abcam, UK) as loading control.
2.9. Statistical analyses Each point corresponds to the mean ± SEM. Statistical differences were determined using the one-way analysis of variance, and p < 0.05 was considered significant. Individual groups were compared using the unpaired Student’s t test.
2.3. Total cell count and differential cell count The bronchoalveolar lavage was done in the left lung. Bronchoalveolar lavage fluid (BALF) samples were centrifuged, and the pellet was resuspended in PBS. The total number of nucleated cells in BALF was counted with a hemocytometer. Then the resuspended BALF was centrifuged onto slides and stained with Wright–Giemsa stain. The slides were used to quantify neutrophil number by counting a total of 200 cells per slide. 2.4. Protein concentration and cytokine levels in BALF BALF protein concentration was measured using bicinchoninic acid protein assay (Thermo Fisher Scientific, MA, USA). Tumor necrosis factor-␣ (TNF-␣), interleukin-1 (IL-1), interleukin-6 (IL6), and macrophage inflammatory protein-2 (MIP-2) levels in BALF were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA) according to manufacture’s guideline. 2.5. Lung morphometry analyses The lungs were fixed in 10% formalin, and 5 m sections were cut for hematoxylin and eosin staining. Lung injury was scored according to the following variables: alveolar and interstitial edema, neutrophil infiltration and hemorrhage. Each variable
3. Results 3.1. KGF-2 time-dependent protective effects on LPS-induced ALI Intratracheal instillation of LPS induced ALI which is represented by a significant decrease in arterial PO2 , and a significant increase in BALF protein concentration, BALF total cell number, BALF neutrophil number, lung wet-to-dry weight ratio, and lung injury score 24 h after LPS administration compared to that in rats receiving PBS as control (Fig. 1). In order to define the best response timing for rhKGF-2 on lung injury prevention, rhKGF-2 was instilled at different time points. Indeed, intratracheal instillation of rhKGF-2 1–5 days before LPS challenge showed different improvements in lung injury compared to animals without pretreatment (Fig. 1). Pretreatment with rhKGF2 two or three days before LPS challenge resulted in a significant improvement in lung injury parameters compared with that in control rats receiving no pretreatment. Although there is no significant difference of above parameters between 2 and 3 days before LPS instillation, absolute number indicates pretreatment 3 days prior to insult might be slightly more effective than the other time points. So we decided to further investigate the protective effect of rhKGF-2 pretreatment 3 days before challenge in LPS-induced ALI.
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Table 1 Primer sequences for quantitative real-time polymerase chain reaction. Genes
Primers (5 –3 )
GenBank accession no.
Amplicon size (bp)
Interleukin-1
F-GCTGTGGCAGCTACCTATGTCTTGR-AGGTCGTCATCATCCCACGAGF-CCACTTCACAAGTCGGAGGCTTAR-GTGCATCATCGCTGTTCATACAATCF-AGGAGCTCCAGACTGAACTCTATGAR-CTGACTGCCCATTGGTGGAAF-GGGCCTTCACATGAGTCAGAAACR-CGATGCCAGTGGAGCCAATAF-GGAGATTACTGCCCTGGCTCCTAR-GACTCATCGTACTCCTGCTTGCTG-
NM 031512
120
NM 012589.1
108
NM 017329
123
NM 017342
140
NM 031144.2
150
Interleukin-6 Surfactant protein A Surfactant protein C -Actin
3.2. Cytokines and pathological changes with KGF-2 pretreatment 3 days before LPS challenge RhKGF-2 was administrated three days before LPS challenge. Four groups of rats, with/without LPS and with/without rhKGF-2 were compared to each other for lung injury measurement. LPS instillation significantly increased the levels of TNF-␣, IL-1, IL-6, and MIP-2 in BALF, while pretreatment of rhKGF-2 significantly reduced the levels of these cytokines (Fig. 2). The mRNA expression of IL-1 and IL-6 in lung tissue was similarly reduced with rhKGF2 (Fig. 2). Representative histological features of LPS-induced ALI were protein-rich edema fluid in alveolar space, leukocytes accumulation, hemorrhage and bronchial wall thickening with
inflammatory cells. Those changes were improved by rhKGF-2 pretreatment, and resulted in a lower score in lung injury than the vehicle pretreated groups (Fig. 3). 3.3. The change of surfactant proteins It was shown that the mRNA expression levels of SP-A and surfactant protein C (SP-C) were significantly decreased with LPS instillation, while rhKGF-2 pretreatment three days before partially restored the expression of SP-A and SP-C (Fig. 4). The protein expression level was measured, and the similar pattern was shown as the mRNA level (Fig. 4). Notably, rhKGF-2 administration alone increased the expression of SP-A and SP-C.
Fig. 1. Effect of rhKGF-2 on lung injury indices following intratracheal LPS. RhKGF-2 was adminstrated 1 (1d), 2 (2d), 3 (3d), or 5 (5d) days before LPS challenge, and no pretreatment was given to the control group (challenged with PBS instead of LPS) and LPS group(receiving 5 mg/kg LPS only). The effects of rhKGF-2 pretreatment on arterial PO2 (PaO2 ) (A), BALF total cell count (B) and neutrophil count (C), BALF protein concentration (D), lung wet-to-dry weight ratio (E), and lung injury score (F) following intratracheal LPS were evaluated. Data are expressed as mean ± SEM (n = 4 for each group). *p < 0.05, **p < 0.01 compared with control, and †p < 0.05, ††p < 0.01 compared with the LPS group.
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Fig. 2. The levels of cytokines in bronchoalveolar lavage fluid (BALF) and lung tissue. Pretreatment with rhKGF-2 markedly reduced the levels of tumor necrosis factor-␣ (TNF-␣) (A), macrophage inflammatory protein-2 (MIP-2) (B), interleukin (IL)-1 (C) and IL-6 (D) in BALF and the mRNA expression levels of IL-1 (E) and IL-6 (F) in lung tissue. Data are expressed as mean ± SEM (n = 6 for each group). *p < 0.05, **p < 0.01 compared with control, and †p < 0.05, ††p < 0.01 compared with the LPS group.
3.4. The proliferation of alveolar type II epithelial cells 2–6 days after KGF-2 treatment
hyperplastic alveolar epithelial cells contained one or more lamellar inclusions (Fig. 5).
Immunohistochemistry staining was conducted for proSP-C, which is expressed solely on alveolar type II epithelial cells, and Ki67, a proliferation marker of cells. RhKGF-2 was injected intratracheally at a single dose of 5 mg/kg, and the lungs at 2, 3, 4, 6 days after injection were harvested. Immunoreactive proSP-C was demonstrable in hyperplastic alveolar lining cells 2 days after rhKGF-2 treatment. The number of proSP-C positive cells in rhKGF2 treated lungs was one-fold increased at day 2 compared to vehicle treated lungs, then slightly decreased and normalized at day 6 (Fig. 5). Ki67 expression was prominently detected in alveolar type II epithelial cells at 2 day after the intratracheal injection of rhKGF-2, a time point at which epithelial cell hyperplasia was morphologically recognizable. And the Ki67 expression was not prominent at day 6. The type II pneumocytes in the lungs of control rats showed only rare expression of Ki67. Meanwhile, rhKGF-2 also caused an increase of Ki67 expression in bronchiolar airways that was more focal and less striking than that in the alveolar type II epithelial cells (Fig. 5). The lungs of rhKGF-2 treated rats were ultrastructurally examined 2 days after intratracheal injection, and it showed that the
4. Discussion We have shown that intratracheal administration of rhKGF2 at a dose of 5 mg/kg body weight two or three days prior to insult had significant protective effects on LPS-induced ALI in rats. Additional studies showed that pretreatment of rhKGF-2 partially restored lung SP-A and SP-C expression after LPS challenge. Our study showed that there was proliferation of alveolar type II epithelial cells in lung parenchyma after rhKGF-2 instillation. These data demonstrate that KGF-2 pretreatment can ameliorate the LPSinduced ALI effectively. A series of studies have demonstrated that KGF has potential protective effects in ALI induced by various stresses (FrancoMontoya et al., 2009; Ulrich et al., 2005; Terry et al., 2004; Welsh et al., 2000; Viget et al., 2000; Yano et al., 1996; Sugahara et al., 1998). Due to its huge similarities to KGF (Igarashi et al., 1998), KGF2 was supposed to be even more potent in protection of lung injury because unlike KGF, KGF-2 receptor was expressed on both epithelial and endothelial cells (Huang and Berkland, 2009; She et al., 2012). Gupte et al. (2009) recently reported the anti-fibrotic effect of KGF-2 on bleomycin-induced pulmonary fibrosis. Our group also
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Fig. 3. Histological quantitation of lung injury following LPS challenge. Sections of hematoxylin and eosin-stained lung tissue were examined under light microscopy (5×), and damage was recorded using the parameters described in Section 2. (A) Section from control animals receiving intratracheal PBS showing alveolar spaces free of inflammatory cell infiltration, with no evidence of hemorrhage or edema. (B) Pretreatment of rhKGF-2 without LPS challenge showed similar appearance as the lung of (A). (C) LPS administration (5 mg/kg) results in the appearance of edematous areas, with a large number of inflammatory cells infiltration. (D) Pretreatment of rhKGF-2 ameliorates lung damage following LPS instillation. Sections are representative of 6 animals examined per group.
showed significant improvement in survival and lung injury in a high altitude pulmonary edema rat model with protection on epithelium and endothelium stress (She et al., 2012). The mechanisms underlying the protective effect of KGF-2 in ALI especially LPS-induced ALI remain incompletely understood
but are probably multiple. The present study investigated the role of KGF-2 on lung inflammation and permeability caused by LPS instillation, the mitogenic effect on alveolar type II epithelial cells and changes in the production of surfactant proteins by epithelial cells.
Fig. 4. The mRNA and protein expression levels of surfactant protein A (SP-A) and surfactant protein C (SP-C) in lung tissue. (A) Relative mRNA expression level of SP-A in lung tissue. (B) Relative mRNA expression level of SP-C in lung tissue. (C) Western blot analysis of SP-A expression level in lung tissue. (D) Western blot analysis of prosurfactant protein C (proSP-C) expression level in lung tissue. Data are expressed as mean ± SEM (n = 6 for each group). **p < 0.01 compared with control, ††p < 0.01 compared with the LPS group.
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Fig. 5. KGF-2 stimulates alveolar type II epithelial cell proliferation. Labeling was obtained by incubation with anti-prosurfactant protein C (proSP-C) antibody (A) or anti-Ki67 antibody (B–D). Sections were covered with DAB tetrahydroxychloride, and counterstained with hematoxylin. (A) Immunoreactive proSP-C is demonstrable in hyperplastic alveolar lining cells 2 days after intratracheal injection of 5 mg/kg rhKGF-2 (400×). (B) Ki67 expression was prominently detected in alveolar type II epithelial cells 2 days after the intratracheal injection of rhKGF-2 (400×). (C) The alveolar type II epithelial cells in the lungs of control rats do not demonstrate any Ki67 expression in many microscopic fields, although occasional alveolar epithelial cells did express Ki67 (400×). (D) RhKGF-2 causes an increase in Ki67 expression in bronchiolar airway epithelium 2 days after intratracheal injection of rhKGF-2 (200×). (E) An electron micrograph (6000×) shows a group of hyperplastic alveolar epithelial cells containing cytoplasmic lamellar inclusions 2 days after intratracheal injection of rhKGF-2. The lamellar structure of the inclusions is appreciated at higher magnification (20,000×).
4.1. KGF-2 attenuates the inflammatory reaction in LPS-induced ALI Intratracheal instillation of LPS induces a rapid influx of neutrophils into lungs. This study consistently demonstrated that LPS administration significantly increased the neutrophil number in
BALF, while KGF-2 pretreatment markedly reduced the increase of neutrophil number in BALF, suggesting that KGF-2 pretreatment attenuates LPS-induced lung inflammation at least partly through reduced recruitment of neutrophils. Some cytokines play key roles in the initiation and development of lung inflammation including TNF-␣, IL-1, IL-6, and MIP-2
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(Strieter et al., 1999). LPS has been proved to induce secretion of these cytokines in vivo and in vitro (Su et al., 2004b; Suzuki et al., 2000). In this study, TNF-␣, IL-1, IL-6 and MIP-2 levels were markedly decreased in rats with KGF-2 pretreatment. Thus it suggests that KGF-2 pretreatment may suppress LPS-induced lung inflammation through inhibited production of cytokines and the consequent reduction of neutrophil infiltration. 4.2. KGF-2 reduces lung epithelial permeability after LPS challenge Intratracheal instillation of LPS has been shown to increase pulmonary endothelial and epithelial permeability, resulting in pulmonary edema (Su et al., 2004b). We recently found that KGF-2 pretreatment significantly reduced the hypoxia-induced increase in endothelial permeability both in vitro and in vivo via downregulation of apoptosis (She et al., 2012), while in this study, KGF-2 pretreatment attenuated the LPS-induced increases in BALF total protein level and lung wet-to-dry weight ratio, suggesting the protective effects of KGF-2 on lung epithelial barrier. 4.3. KGF-2 stimulates alveolar type II epithelial cell proliferation It has been reported that KGF is a growth factor for alveolar type II epithelial cells in vivo and in vitro (Ulich et al., 1994; Morikawa et al., 2000), and the maximum paracrine mitogenic effects of KGF on epithelium was 2–3 days after pretreatment (Ulich et al., 1994). However, the role of exogenous KGF-2 in alveolar epithelial proliferation has not been reported yet. Our data suggests that there is a substantial increased number of alveolar type II epithelial cells in lung parenchyma 2 days after KGF-2 treatment, and the extent of alveolar type II epithelial cell hyperplasia was similar to that triggered by KGF. The histologic manifestation of the alveolar epithelium on day 6 is much similar to the appearance of normal lungs, suggesting temporal changes of alveolar type II epithelial cells after KGF-2 pretreatment and its potential safety in future clinical implications. 4.4. KGF-2 restores SP- A and SP-C synthesis after LPS stimulation Surfactant is a complex mixture of lipid and protein, secreted by alveolar type II epithelial cells, and regulates surface tension at the pulmonary air–liquid interface to prevent alveolar collapse (Whitsett and Weaver, 2002). Thus we examined the expression of surfactant proteins in lung tissue and demonstrated that KGF-2 pretreatment partially restored SP-A and SP-C expression. However, there was no remarkable change of SP-B before and after KGF-2 treatment (data not shown). The increased expression of surfactant proteins might be due to alveolar type II epithelial cell proliferation and differentiation, and the change of intracellular surfactant proteins per single cell needs to be further addressed. This increase in surfactant proteins production might indirectly prevent a permeability defect, and might be a potential mechanism for KGF-2. Lung epithelial and endothelial repair play important roles in recovery of ALI. Recent studies have shown that stem cells promote lung injury recover through parasecretion of KGF (Lee et al., 2009). Both exogenous KGF and KGF gene delivery via mesenchymal stem cells can protect against LPS-induced ALI (Chen et al., 2013; Hu et al., 2010). The clinical application of KGF has been brought to frontier, since KGF has presently planned clinical randomized controlled trials in ALI to reduce pulmonary dysfunction (Cross et al., 2013). Our studies support the potential use of KGF-2 in prophylactic treatment of ALI. Intratracheal administration of KGF-2 in clinical scenario might be a good safe strategy, because topical application can reduce the side effects of systemic use. KGF-2 given 2–3 days before LPS challenge showed maximum lung injury improvement.
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This phenomenon suggests minimum of 2 days is need to allow KGF-2 reaching full effects and the duration of KGF-2 is also short which can be used in disease treatment without too much concern of the safety due to quick metabolism. Although in this study the data support the prophylactic use of KGF-2 in ALI induced by infectious insults, it should be noted that the animal model of ALI is of much limitation. This ALI model is an acute model, the lung injury recovers or deteriorates within several days. Thus when applying KGF-2 after lung injury, the animals recover or deteriorate before KGF-2 could reach its best effect. This might be the key reason why KGF-2 application after injury could not show marked improvement in lung injury. But this is quite different with bedside situation, because patients of ALI/ARDS experience much longer duration of disease. There might be enough time for KGF-2 to exert its multiple functions on modifying lung homeostasis to promote the recovery from ALI. However, due to the differences between humans and animals, the effects and mechanisms of KGF-2 in alveolar epithelium-capillary endothelium barrier repair after lung injury still needs further investigation. The possibility that KGF-2 may initiate excessive fibrosis after ALI should be a concern when considering the clinical use of KGF2. She et al. (2012) observed the long-term (1 and 2 months) effects of KGF-2 on multiple organs including lung, brain, liver, heart and kidney, and demonstrated that the tissues of those organs were normal and without excessive fibrosis. Furthermore, previous study showed that overexpression of KGF-2 during both inflammatory and fibrotic phases attenuated bleomycin-induced pulmonary fibrosis in mice (Gupte et al., 2009). Therefore, it seems that KGF-2 is a safe agent for treating patients with ALI/ARDS. However, it still needs to be more thoroughly studied as to the long-term effects in clinical trials for safety data in patients and to combinations with other agents used in ALI/ARDS, such as surfactant instillations and steroids. In conclusion, our study demonstrates the protective effect of KGF-2 on LPS-induced ALI, which may be mediated by reduction of pro-inflammatory cytokine levels and inflammatory cell infiltration, restored synthesis of SP-A and SP-C, and proliferation of alveolar type II epithelial cells. Therefore, KGF-2 may have potential clinical application as a prophylactic treatment of ALI. Acknowledgments We acknowledge Shanghai Newsummit Biopharma Co. for generously supplying rhKGF-2, Zude Xu, Shaohua Lu and Haiying Zeng (Fudan University) for the assistance in histological analyses. This research was supported by Shanghai Leading Academic Discipline Project (B115), and National Natural Science Foundation of China (30930090, 81100046, 81170056). Dr. Yuanlin Song was supported by the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning and Key Medical grant from Shanghai Science and Technology Committee (11411951102, 12JC1402300). References Alderson, R., Gohari-Fritsch, S., Olsen, H., Roschke, V., Vance, C., Connolly, K., 2002. In vitro and in vivo effects of repifermin (keratinocyte growth factor-2 KGF-2) on human carcinoma cells. Cancer Chemother. Pharmacol. 50, 202–212. Beer, H.D., Vindevoghel, L., Gait, M.J., Revest, J.M., Duan, D.R., Mason, I., Dickson, C., Werner, S., 2000. Fibroblast growth factor (FGF) receptor 1-IIIb is a naturally occurring functional receptor for FGFs that is preferentially expressed in the skin and the brain. J. Biol. Chem. 275, 16091–16097. Benjamin, J.T., Smith, R.J., Halloran, B.A., Day, T.J., Kelly, D.R., Prince, L.S., 2007. FGF10 is decreased in bronchopulmonary dysplasia and suppressed by Toll-like receptor activation. Am. J. Physiol. Lung Cell Mol. Physiol. 292, L550–L558. Chang, H.L., Sugimoto, Y., Liu, S., Wang, L.S., Huang, Y.W., Ye, W., Lin, Y.C., 2009. Keratinocyte growth factor (KGF) regulates estrogen receptor-alpha (ER-alpha) expression and cell apoptosis via phosphatidylinositol 3-kinase (PI3K)/Akt pathway in human breast cancer cells. Anticancer Res. 29, 3195–3205.
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