PTD-FGF2 Attenuates Elastase Induced Emphysema in Mice and Alveolar Epithelial Cell Injury

Abstract Aberrant communication in alveolar epithelium is a major feature of inflammatory response for the airway remodeling leading to chronic obstructive pulmonary disease (COPD). In this study, we investigated the effect of protein transduction domains (PTD) conjugated Basic Fibroblast Growth Factor (FGF2) (PTD-FGF2) in response to cigarette smoke extract (CSE) in MLE-12 cells and porcine pancreatic elastase (PPE)-induced emphysematous mice. When PPE-induced mice were intraperitoneally treated with 0.1–0.5 mg/kg PTD-FGF2 or FGF2, the linear intercept, infiltration of inflammatory cells into alveoli and pro-inflammatory cytokines were significantly decreased. In western blot analysis, phosphorylated protein levels of c-Jun N-terminal Kinase 1/2 (JNK1/2), extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinases (MAPK) were decreased in PPE-induced mice treated PTD-FGF2. In MLE-12 cells, PTD-FGF2 treatment decreased reactive oxygen species (ROS) production and further decreased Interleukin-6 (IL-6) and IL-1b cytokines in response to CSE. In addition, phosphorylated protein levels of ERK1/2, JNK1/2 and p38 MAPK were reduced. We next determined microRNA expression in the isolated exosomes of MLE-12 cells. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, level of let-7c miRNA was significantly increased while levels of miR-9 and miR-155 were decreased in response to CSE. These data suggest that PTD-FGF2 treatment plays a protective role in regulation of let-7c, miR-9 and miR-155 miRNA expressions and MAPK signaling pathways in CSE-induced MLE-12 cells and PPE-induced emphysematous mice.


Introduction
Chronic obstructive pulmonary disease (COPD) is characterized by excessive formation of mucus and airway structural changes (chronic bronchitis) and inordinate enlargement and destruction of the alveolar region (emphysema) [1,2].Moreover, cigarette smoke and environmental pollutants are considered as main risk factors for COPD [3].In the lung, alveolar epithelium is considered not only essential for pulmonary gas exchange but also functional barrier against a broad variety of stimuli.In the alveolar epithelium, alveolar type II epithelial cells (AT2) are known for the functions in synthesizing and secreting alveoli surfactant and have multiple functions including self-renewal, removing injured AT2 cells, proliferation, and cell-differentiation into AT1 cells for alveolar regeneration [4,5].Repeated exposure of toxic substances to AT2 cells resulted in development of protease-antiprotease imbalance alveolar damage such as imbalance between neutrophil elastase and alpha-1-antitrypsin [6,7], and they subsequently acquire remarkable reparative capacity to release the secretome and chemotactic substances across the capillary-alveolar barrier after injury.Persistent imbalance of a protease-antiprotease consequently repeated a vicious cycle of inflammation and parenchymal remodeling [8][9][10].The paracrine cell-to-cell communication of AT cells is recognized as a major mechanism underlying the physiological homeostasis in the airway remodeling [11][12][13].However, it is remained which the molecular signals modulate in cell-to-cell networks.
Fibroblast growth factor (FGF) family comprises 18 mammalian members which are grouped into seven subfamilies.The FGFs are implicated in a wide range of biological processes including organogenesis homeostasis and pathogenesis of respiratory diseases [14].In FGF family, FGF2 (basic FGF or FGF2) exhibited the pivotal role in the various diseases such as major depressive disorder (MDD) [15], periodontitis [14], and coronary artery disease [16].In the lung, FGF2, a potent mitogen, has been reported to be expressed in the smokers with chronic bronchitis.In addition, increased level of FGF2 recognized a relevant remodeling biomarker of asthma severity and it was significantly associated with pulmonary function [17].On the other hand, FGF2 plays an essential role in cell proliferation and apoptosis of airway smooth muscle migration, airway hyper-responsiveness (AHR), mucus production as well as epithelial repair [18,19].In previous trials with FGF2 treatment in COPD experimental models, gelatin-embedded, sustained-release FGF2 has been administrated intra-arterially in rats, and the administration induced lung regeneration with elastase-induced pulmonary emphysema.Kawago and colleagues demonstrated its beneficial application with the sustained-release materials, however, they reported the limited route through artery as well as the unknown mechanism in the lungs [20].
It has been reported that small cationic peptides, called protein transduction domains (PTDs), efficiently deliver the large, bioactive molecules into the cells across cellular membrane [21].PTDs are referred as to cell-penetrating peptides have been developed for delivery of therapeutic protein into the target cells in various diseases.In ovalbumin-induced allergic asthma mice, human transcriptional factor-1 fused into cytoplasmic domain of CTLA-4 treatment prevented the infiltration of inflammatory cells suppressing the development of bronchial asthma [22].In addition, PTD-BDNF fusion protein promoted the growth of hippocampal neurons against Aβ induced toxicity and transported through blood-brain barrier in mice [23].In peritoneal dialysis of rats, PTD-BMP7 treatment decreased EMT-related fibrosis to reduce peritoneal membrane damage [24].Although numerous experimental studies suggest therapeutic intervention to upregulate repair of damaged alveolar epithelial cells, it is still remained how injured alveolar epithelial cells participate in the pathogenesis of COPD, and beneficial effect of AT2 exosomes by PTD-FGF2 to promoting survival and proliferation.Therefore, in this study, we demonstrate the identified a novel factor with PTD-FGF2 and its therapeutic role in exosomal secretion of AT2 cells and lung tissues after injury.

Animal experiment and ethics statement
8 weeks old male C57BL/6 mice were purchased by Doo Yeol Biotech (Seoul, Republic of Korea).All procedures were approved by the Institutional Animal Care and Use Committee (Permit Number: KW-150820-1) at Kangwon National University, Republic of Korea, and whenever necessary, adequate anesthetic regimen was used to minimize suffering.For animal experiments, mice were allowed to acclimatize for a minimum period of 7 days in an animal facility, during which time they were allowed free access to water and standard chow.

Lung harvest, Bronchoalveolar lavage fluid (BALF) isolation, and cell counts
Right lungs were homogenized with a tissue homogenizer in Trizol (#79306, QIAGEN, NRW, Germany) and RIPA buffer.Left lungs were fixed by 4% paraformaldehyde, embedded in paraffin, and cut into 6 μm thick sections.Airspace enlargement was evaluated by measurement technique of the mean linear intercept (MLI).To measure the intercepts, representative photomicrographs of lung parenchyma with H&E staining were randomly selected, and the interalveolar septal wall distance was measured using ToupView software on computer connected with microscopy (n = 3 images per groups) as described by G Crowley et al. [25].BALF was centrifuged at 3,000 rpm, 10 min, 4 °C and the isolated cells were attached on slide in cytospin centrifuge at 3,000 rpm for 5 min.The attached cells were stained by Hema-3 staining kit (Thermo Fisher Scientific), and evaluated under the microscope (magnification, x200).

Measurement of intracellular reactive oxygen species (ROS) and nitrogen species (RNS)
The level of intracellular ROS was detected with 2′, 7;-dichlorofluorescein-diacetate (DCF-DA).The treated cells were loaded with 10 μM DCF-DA for 30 min at 37 °C.Then, cells were dissociated with 0.25% trypsin and analyzed by flow cytometer (Accuri C6, BD Biosciences, CA, USA).To determine the level of nitric oxide, RNS was determined by modified Griess (#G4410, SIGMA-ALDRICH, MO, USA) methods.The absorbance was measured at 540 nm using microplate reader.

Immunostaining
MLE-12 cells and lung tissues were fixed by 4% paraformaldehyde for 10 min, permeabilized with Triton X-100 for 10 min, blocked with 10% normal goat serum for 30 min, and stained with anti-His probe antibody combination with anti-rabbit IgG (Alexa Fluor 488 conjugate, #4412, Cell signaling).The slides were mounted by Fluoroshield with DAPI (#6057, SIGMA-ALDRICH, MO, USA) and analyzed under the confocal microscope.

Exosome isolation and quantification
The MLE-12 cells were grown for up to 48 h in serum-free culture medium based on DMEM/F12 including 1% penicillin-streptomycin supplemented with 0.005 mg/mL insulin, 30 μM sodium selenite, 10 nM hydrocortisone, and 10 nM β-estradiol at 37 °C and 5% CO 2 to avoid contaminate cell derived exosomes from the FBS-derived exosomes.To isolate exosome in MLE12 cells, the cells were treated with FGF2, PTD-FGF2 and/or CSE.After 24h, the cell conditioned culture medium (CM) was collected in 50 mL Falcon tube, centrifuged to remove cell debris in 2,000 rpm C for 10 min, and eliminated additional contamination in 10,000 rpm at 4 °C for 30 min.The supernatant was filtered by 0.2 μm sterile filter (#16534, Sartorius stedim, Germany), and ultracentrifuged at 75,000 g [1,26].The isolated exosome was measured by the FluoroCet Ultrasensitive Exosome Quantitation Assay kit (SBI, USA) according to manufacturer's instructions and absorbance.

Nanoparticle tracking analysis
The supernatants were diluted in PBS to obtain a concentration within the recommended measurement range (1-10 × 10 8 particles/mL) [27].The isolated nanoparticles were measured by NanoSight instruments (NS300, Malvern Panalytical, United Kingdom), and analyzed with NTA 2.3 software.

Statistical analysis
The statistical results were analyzed by GraphPad Prism 5.0 (GraphPad Software, CA, USA).Each result was tested using One-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test.A value of *p < 0.05, **p < 0.01, ***p < 0.001 was compared control for all pair.All experiments were performed at least three times.

Effect of PTD-FGF2 in PPE-induced emphysematous mice
To determine whether PTD-FGF2 treatment attenuates emphysema development, mice received PPE were treated with FGF2 or PTD-FGF2.Intratracheal PPE instillation caused significant increase in airspace enlargements and alveolar wall destruction.Treatment with FGF2 significantly suppressed the increases in mean linear intercept (Lm), and PTD-FGF2 treatment further inhibited the increase of Lm values observed in mice treated with PPE (Figure 1A and B).Fourteen days after PPE instillation, increased number of alveolar neutrophil, macrophage and lymphocyte were recruited into lung airspaces.Rather FGF2 and PTD-FGF2 treated group exhibited an altered inflammatory response to elastase over time.The elevated numbers of lavagable neutrophil, macrophage and lymphocytes were significantly inhibited in FGF2 and PTD-FGF treated groups (Figure 1C-F).Furthermore, PPE instillation increased statistically the level of pro-inflammatory cytokines including TNF-α, IL-6, and IL-1β, which was effectively blocked by both FGF2 and PTD-FGF2 by a similar level (Figure 2A-C).We next evaluated the effect of PTD-FGF2 on phosphorylation of three MAPKs including ERK1/2, p38 and JNK1/2 in the lungs of PPE-exposed mice.FGF2 treatment attenuated the MAPK pathway and PTD-FGF2 treatment further inhibited on phosphorylations of ERK1/2, p38 and JNK1/2 (Figure 2D-E).These findings suggest that PTD-FGF2 rather than FGF2 treatment significantly inhibits infiltration of inflammatory cells and pro-inflammatory cytokine secretion through MAPK signaling in PPE-induced emphysematous changes.

PTD-FGF2 treatment induces the release of extracellular vesicles
We next determined the immunocytochemical distribution of PTD-FGF2 conjugated by His protein in MIL-12 cells and mouse lung tissues.As shown in Figure 4A, fluorescence of His in PTD-FGF2-treated MLE-12 cells was dispersed widely in cytoplasm, and Figure 4B displayed strong intensities of green florescence in lung alveoli of PTD-FGF2injected mouse.We next determined whether PTD-FGF2 treatment induces more secreting exosomes rather than FGF2 treatment in MLE-12 cells.The exosomes were isolated and purified from PTD-FGF2 or FGF2 treated MLE-12 cells using a standard protocol described previously.Electron microscopy analysis revealed exosomes were typical spheroids or cup-shaped particles (Figure 4C).PTD-FGF2 treatment significantly increased total concentration of particles, exosome quantity and size distribution rather than FGF2 treatment in MLE-12 cells (Figure 4D-F).Nanoparticle tracking analysis confirmed that the particle size of the MLE-12 derived exosomes mainly ranged from 50 to 200 nm (Figure 4F).In addition, isolated exosomes from MLE-12 cells and mouse BALF did not exhibit protein level of cytochrome C (Cyto C) that are readily detectable in the whole cell lysates, whereas protein levels of HSP70, TSG101, CD9 and CD63 as exosomal markers were expressed (Figure 4G).These data suggest that PTD-FGF2 treatment is capable of reaching intercellular compartments of AT2 cells without disruption of the cell membrane and mouse lung tissues leading to induce the release of exosomes.

PTD-FGF2 treatment regulates the exosomal microRNAs
PTD-FGF2 treatment have stimulated the exosomal secretion of AT2 and lung tissues in this study.Next, we determined the expression level of several exosomal microRNAs (miRNA) in AT2 cells and whole lung homogenates, because exosomal miRNAs are the dominant exosomal cargo molecules [32].Accordingly, MLE-12 cells derived exosomes were isolated and expression of microRNAs were determined by RT-PCR analysis.The level of endogenous Let-7c was significantly decreased in response to CSE, whereas PTD-FGF2 treatment restored the Let-7c levels (Figure 5A).Moreover, the increased level of Let-7c treated with FGF2 and PTD-FGF2 has determined in lungs of PPE exposed mice (Figure 5D).These results suggested that PTD-FGF2 treatment effectively leads to the delivery of Let-7c by exosome secretion on injured AT2 cells.In contrast, the levels of endogenous miR-9 and miR-155 which play a central role in inflammatory response were significantly increased in CSE exposed MLE-12 cells and PTD-FGF2 treatment significantly inhibited the upregulation of both miR-9 and miR-155 (Figure 5B-C).In mice lungs treated with PPE, the miR-9 and miR-155 were highly expressed, whereas PTD-FGF2 treatment decreased the levels of both miR-9 and mir-155 of lung homogenates (Figure 5E-F).These results suggested that PTD-FGF2 treatment effectively leads to the exosome-mediated restoration against excessive expression of endogenous miR-9 and miR-155 on inflammation of AT2 cells.

Discussion
COPD is a heterogeneous disease including both alveolar and airway compartments, and cigarette smoke (CS) in external risk factors representatively induces alveolar damage leading to remodeling of lungs [2].AT2 dysfunction and dropout by cigarette smoke has been reported in mammalian epithelial cells and COPD experimental mice including our previous studies [3,4].Recently, extracellular vesicles (EVs) which are surrounded by phospholipid bilayer are emerging as the functional factor in cell-to-cell communication [5].The exosomes via endo-lysosomal pathway include messenger RNA (mRNA), micro RNA (miRNA), membrane protein, and EVs are implicated in delivering their component from therapeutic agents or damaged cell to other cells [14].In the lung, EVs exist in bronchoalveolar lavage (BAL) fluid, and released exosomes by the respiratory composed cells including T-lymphocytes, mast cells, dendritic cells and epithelial cells, have been suggested to involve antigen delivery or immune regulation during airway antigen exposure [15].Moreover, the clinical data in COPD patients with healthy smokers and nonsmokers demonstrated the EVs and the level of miRNAs are involved in the pathogenesis and diagnosis of COPD [33,34].Despite EVs' participation in the physiology and disease progression, it is unknown how exosome secretion by AT2 cells plays a role in airway microenvironment.PTD fused proteins to EGF and FGF1 treatments have been applied with skin injury and ameliorated the UV-induced skin damage [16].The PTD-EGF1 and FGF1 proteins efficiently transduced into skin cells and increased cell proliferation via up-regulation of MAPK signaling [16].In this study, PTD-FGF2 treatment has been applied to CSE exposed MLE-12 cells, and they exhibited decreased release of TNF-α, IL-6, IL-1β as well as production of NO and ROS in response to CSE.These data are in accordance with CS caused epithelial cell damage via generation of ROS and RNS [4,19,[35][36][37].Guzy et al. have reported that expression of Sftpc, Cdh1, and Aqp5 were decreased in FGF2 −/− mice after bleomycin suggesting that FGF2 signals directly to recovering alveolar epithelium [20].In interferon-γ induced emphysema mice, recombinant FGF2 treatment inhibited the development of emphysema and non-eosinophilic asthma phenotype [21].Moreover, when beagle dogs intratracheally received PPE, FGF2 treatment functionally relieved pulmonary blood flow and increased feasibility of bronchoscopic lung volume reduction [22].In line with previous studies, PTD-FGF2 has protective effect from CSE-induced AT2 cell injury including decreased proliferation, increased ROS and inflammatory cytokines.
In COPD patients, constitute or aberrant MAPK signaling contributes to pathogenesis of COPD-associated phenotypes [23,[38][39][40].We have reported that up-regulation of ERK1/2, JNK1/2 and p38 MAPK dependent on Nrf2 migration were associated with regulated apoptosis of AT2 cells [3].Accordingly, this report demonstrates the inhibitory contribution of PTD-FGF2 in MAPK signaling activation of CSE-induced AT2 cells and emphysematous mice.The mature miRNAs controls the transcriptional modulation or translational silencing of target mRNA [24,41].Additionally, miRNAs orchestrate physiological and pathological process such as immune responses, organ development, cell proliferation, differentiation and apoptosis [42,43].The microRNAs have emerged as critical targets to determine the phenotypic heterogeneity, and future prognosis and diagnosis of COPD [44][45][46].Fujita et al. determined that human bronchial epithelial cell-derived EVs with CSE were differentiated to myofibroblasts by upregulated miR-210 [5].In addition, CSE exposed lung epithelial-derived EVs promoted from full-length CYR61/CTGF/NOV family1 (flCCN1) to cleaved CCN and increased MMP1 that accelerated emphysematous lung [47].Schembri et al. found 28 miRNAs differentially expressed in smokers through gene expression profiling.Among them, decreased MiR-218 was involved in cell proliferation, apoptosis and cellular stress affected cellular differentiation in CS exposure airway epithelial cells [48].In this study, we found that levels of let-7c, miR-9 and miR-155 are significantly involved in PTD-FGF2 treatment in injured AT2 cells.Several researchers has reported that the strong downregulation of let-7c in the lungs of mice exposed to total particulate matter and in sputum macrophage and airway epithelial cells of COPD patients [49][50][51].We have shown that PTD-FGF2 treated both AT2 cells and lungs of mice were maintained in the level of let-7c in response to CSE or PPE injury.It suggests that PTD-FGF2 treatment led to increased exosomal let-7c release inhibiting cellular damage and oxidative stress.A potential role of miR-155 in the adaptive immune response was provided in immune cells.Moreover, its reduction suppressed IL-8 mRNA and concomitantly reduced expression of IL-8 protein in lung epithelial cells of cystic fibrosis [52,53].In addition, miR-9 is increased in cystic fibrosis cells where it directly regulates anoctamin1 resulting in transmembrane conductance regulator deficiency [54].In this report, PTD-FGF2 treatment significantly suppressed upregulation of miR-155 and miR-9, which are in agreement with earlier studies in inflammatory response or dysfunction of alveolar epithelial cells.Although PTD-FGF2 treatment regulates the differential expression of let-7c, miR-155 and miR-9, the involved mechanisms are presently uncertain.Moreover, at present, the functions of let-7c, miR-155 and miR-9 have not been investigated even though their modulated levels would result in a decrease or increase in expression of target proteins.Therefore, it is needed to determine the major target of microRNAs and functionally correlate to pathogenesis of COPD.

Conclusions
In conclusion, to our knowledge, the current study is the first to propose a novel factor with PTD-FGF2 and its pivotal role in exosomal secretion of AT2 cells and lung tissues.In CSE-exposed AT2 cells and emphysematous mice, PTD-FGF2 treatment decreased inflammatory cytokine release, NO, and ROS production via inhibition of MAPK signaling.Importantly, PTD fused to FGF2 peptide efficiently transduced into AT2 cells, and enhanced the exosomal secretion consequently leading to ameliorate alveolar cell damage and lung injury.Significantly, rapid increases in let-7c treated with PTD-FGF2 is able to explain one of major factors in paracrine effect during clearance of AT2 cells.Therefore, these results suggest that pharmacological targeting of miRNAs might contribute a novel therapeutic approach to the treatment of emphysema.Furthermore, application of PTD-FGF2 might provide the considerable treatment for the regulation of inflammation.

Figure 1 .
Figure 1.PtD-fGf2 treatment inhibits infiltration of inflammatory cells in emphysematous mice.C57bl/6 mice were intratracheally injected 5u of PPe in 50 μℓ of sterile Pbs. after 1 d, fGf2 or PtD-fGf2 were injected intraperitoneally for 5 days.after 14 days, mice were sacrificed and lung tissues were harvested.(a) histological analysis in each group using h&e staining.scale bare = 100 μm (b) mean linear intercept (lm) was measured in alveoli space distance.(C) total cells, (D) neutrophils, (e) macrophages and (f) lymphocytes in balf were measured by cytospin and Giemsa staining analysis.**p < 0.01 and ***p < 0.001 compared with control by one-way anOva.† p < 0.01 and † † † p < 0.001 compared with PPe injection by one-way anOva.

Figure 2 .
Figure 2. maPK pathway regulation of PtD-fGf2 treatment in PPe-induced mice.Pro-inflammatory cytokines including (a) tnf-α, (b) il-6 and (C) il-1β were measured by elisa assay.(D) Protein levels of JnK1/2, erK1/2 and p38 maPK was measured by western blot analysis and the samples in each group were loaded from different two batch.(e) relative intensity ratio of band in each group was determined.***p < 0.001 compared with control by one-way anOva.† † p < 0.05 and † † † p < 0.001 compared with PPe injection by one-way anOva.

Figure 3 .
Figure 3. Protective effect of PtD-fGf2 in Cse-induced alveolar epithelial cell damage.PtD-fGf2 was pretreated for 2 hours, and Cse was exposed for 24 hours.(a) the cell viability was determined by mtt assay.(b) the level of nitric oxide was analyzed by Griess assay.(C-e) the pro-inflammatory cytokines including (C) tnf-α, (D) il-6 and (e) il-1β were measured by elisa assay.(f) intracellular rOs was measured by DCf-Da fluorescence.(G) Protein levels of JnK, erK and p38 maPK was measured by western blot analysis.**p < 0.05, ***p < 0.001 compared with control by one-way anOva.† p < 0.05, † † p < 0.01 and † † † p < 0.001 compared with 0.5% Cse treatment by one-way anOva.

Figure 4 .
Figure 4. PtD-fGf2 treatment enhances exosome biogenesis in mle-12 cells and mice.mice and cells were treated with PtD-fGf2 at each concentration.PtD-fGf2 tagged with his was detected by his antibody in (a) mle-12 cells and (b) mouse lung tissue.scale bar = 100 μm (C) exosomes are nanosized vesicles generated by release of mle-12 cells using transmission electron microscopy (sem).scale bar = 100 nm (D) total concentration of mle-12-derived particles was measured by nanoparticle tracking analysis.(e) total exosome quantity of mle-12 was measured by fluocet exosome quantitative kit.(f) the particle size of the exosome in mle-12 was measured by nanoparticle tracking analysis.D10, D50, D70 and D90 mean particle size of distribution, which percentages 10%, 50%, 70% and 90% of particles under the reported particle size.(G) the exosome markers including hsP70, tsG101, CD9 and CD63 were measured by western blot in both mle-12 and mouse balfs.Cytochrome C as mitochondrial marker, was measured the same method.*p < 0.01 and **p < 0.05 compared with control by one-way anOva.† p < 0.05, † † p < 0.01 compared with 0.5% Cse treat by one-way anOva.