Am J Physiol Lung Cell Mol Physiol 307: L718–L726, 2014. First published August 29, 2014; doi:10.1152/ajplung.00293.2013.
Liver growth factor treatment reverses emphysema previously established in a cigarette smoke exposure mouse model Sandra Pérez-Rial, Laura del Puerto-Nevado, Álvaro Girón-Martínez, Raúl Terrón-Expósito, Juan J. Díaz-Gil, Nicolás González-Mangado, and Germán Peces-Barba Respiratory Research Group, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz-CIBERES (IIS-FJD-CIBERES), Madrid, Spain Submitted 15 October 2013; accepted in final form 28 August 2014
Pérez-Rial S, del Puerto-Nevado L, Girón-Martínez Á, TerrónExpósito R, Díaz-Gil JJ, González-Mangado N, Peces-Barba G. Liver growth factor treatment reverses emphysema previously established in a cigarette smoke exposure mouse model. Am J Physiol Lung Cell Mol Physiol 307: L718 –L726, 2014. First published August 29, 2014; doi:10.1152/ajplung.00293.2013.—Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease largely associated with cigarette smoke exposure (CSE) and characterized by pulmonary and extrapulmonary manifestations, including systemic inflammation. Liver growth factor (LGF) is an albumin-bilirubin complex with demonstrated antifibrotic, antioxidant, and antihypertensive actions even at extrahepatic sites. We aimed to determine whether short LGF treatment (1.7 g/mouse ip; 2 times, 2 wk), once the lung damage was established through the chronic CSE, contributes to improvement of the regeneration of damaged lung tissue, reducing systemic inflammation. We studied AKR/J mice, divided into three groups: control (air-exposed), CSE (chronic CSE), and CSE ⫹ LGF (LGF-treated CSE mice). We assessed pulmonary function, morphometric data, and levels of various systemic inflammatory markers to test the LGF regenerative capacity in this system. Our results revealed that the lungs of the CSE animals showed pulmonary emphysema and inflammation, characterized by increased lung compliance, enlargement of alveolar airspaces, systemic inflammation (circulating leukocytes and serum TNF-␣ level), and in vivo lung matrix metalloproteinase activity. LGF treatment was able to reverse all these parameters, decreasing total cell count in bronchoalveolar lavage fluid and T-lymphocyte infiltration in peripheral blood observed in emphysematous mice and reversing the decrease in monocytes observed in chronic CSE mice, and tends to reduce the neutrophil population and serum TNF-␣ level. In conclusion, LGF treatment normalizes the physiological and morphological parameters and levels of various systemic inflammatory biomarkers in a chronic CSE AKR/J model, which may have important pathophysiological and therapeutic implications for subjects with stable COPD. chronic cigarette smoke exposure; emphysema; liver growth factor; mice model; therapy CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) is a major cause of illness and death throughout the world, with the main risk factor as cigarette smoke, and increasing evidence suggests that COPD is a disease characterized by both local and systemic inflammation. Systemic inflammation in COPD is characterized by cytokines implicated in inflammatory cell recruitment and airway remodeling. Reduced lung function is associated with increased levels of various systemic inflammatory markers (16). Emphysema, a component of COPD, is
Address for reprint requests and other correspondence: Sandra Pérez-Rial, Respiratory Research Group, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz-CIBERES (IIS-FJD-CIBERES), Avenida Reyes Católicos, 2, 28040, Madrid, SPAIN (e-mail: [email protected]). L718
characterized by an inflammatory cell infiltrate and by the presence of proteinases in excess within the alveolar space, which leads to destruction of alveolar walls and permanent enlargement of the peripheral airspaces of the lung, and no treatment is currently available to reverse the lung destruction and induce proper healing. The target of newer treatments being developed is primarily directed toward stopping the worsening of the disease, either by inhibiting inflammation and the overproduction of mucus, or by employing antioxidant and antifibrotic agents (3). Liver growth factor (LGF) was identified as an albuminbilirubin complex with mitogenic properties in rat liver (12). It has antifibrotic, antioxidant, and antihypertensive properties, and its regenerative effects have been described in several rodent models, including hepatic damage (13, 14), cardiovascular diseases such as hypertension and atherosclerosis lesions (7, 8, 42), Parkinson’s disease (17, 38, 39), and testicular degeneration (32). On the other hand, data published in lung fibrosis by our group show the antifibrotic effects of LGF in rats induced with ClCd2 (30). Considering the regenerative activity in different systems, LGF could be suggested as a promising choice for the stimulation of processes involved in tissue repair in previously damaged lungs in a known model of COPD in mice. Therefore, our goal is to test the effects of LGF administration in a mouse model of emphysema induced by chronic cigarette smoke exposure (CSE). In several investigations, it has been shown that the development of emphysema in mice chronically exposed to cigarette smoke is highly dependent on the mouse strain and dose (20, 47). In this regard, the AKR/J strain was shown to be extremely susceptible to the development of emphysema, as measured by increases in the airspace enlargement and the elastic properties of the lung, after chronic exposure to cigarette smoke (20). Likewise, the cellular and molecular inflammatory response was also shown to be more prominent in the lungs of the AKR/J mice chronically exposed to cigarette smoke than in the other mouse strains investigated in the study in question (20). Fluorescence molecular imaging (FMI) is the method of choice for functional, real-time, in vivo monitoring of matrix metalloproteinase (MMP) activity because it provides an “easy” approach for measuring enzymatic activities (21, 29, 45). Here we use an injectable pan-MMP-targeted optical sensor that specifically and semiquantitatively resolves MMP-2, -3, -9, and -13 activities that initiate and propagate inflammatory responses (11, 19, 27, 33) in the lungs of mice with experimental chronic CSE-induced emphysema. Several studies have suggested that MMPs are the critical mediators of CSE-induced emphysema (11, 24, 41).
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LGF REVERSES EMPHYSEMA MOUSE MODEL
The purpose of this study was to demonstrate the therapeutic effect of LGF treatment improving the inflammatory status, visualized and quantified by in vivo FMI in the lung of a chronic inflammation after CSE-induced emphysema in AKR/J mouse model, including improvement of lung function and morphometry parameters. MATERIALS AND METHODS
Animals. Thirty adult, 8-wk-old male AKR/J mice, 25–27 g body wt (The Jackson Laboratory, Bar Harbor, ME), were divided in three groups (n ⫽ 10/group): control (CTL), CSE, and CSE with LGF treatment (CSE ⫹ LGF). Animals were housed in the Inhalation Core Facility at IIS-Fundación Jiménez Díaz for 1 wk of acclimatization before the experimental procedures. After daily exposure, mice were returned to their cages, where a diet of alfalfa-free rodent food (Harlan Teklad) and water were provided ad libitum. Protocols were approved by the local Ethical Animal Research Committee at IIS-Fundación Jiménez Díaz. In all cases, the legislation regarding animal treatment, protection, and handling was followed (RD 53/2013). All animals were weighed on a monthly basis during the 6-mo study period. AKR/J mice after chronic CSE for 6 mo exhibited a significantly lesser increase in their body weights compared with the corresponding control animals. Indeed, at 6 mo, mice exposed to tobacco smoke presented a minor increase in their body weight gain, (34% of baseline weight) compared with the CTL group (65% of baseline weight) (4). No significant differences were appreciated in the body weight gain in the group of mice treated with LGF. Chronic CSE-induced emphysema model. Chronic CSE was performed in 20 AKR/J mice (CSE group) with a daily exposition, 5 days a week, during a continuous period of 6 mo. The smoke from two consecutive (with 5-min breaks between them) standard research nonfiltered cigarettes was used (2R1, Cigarette Laboratory at the Tobacco and Health Research Institute, University of Kentucky, Lexington, KY), containing 11.7 mg total particulate matter per cubic meter of air, 9.7 mg tar, and 0.85 mg nicotine per cigarette. Mainstream CSE was generated by an exposure system and was drawn into the chambers via peristaltic pump, following previously published methodologies (4, 33, 35). The whole body of the animals was exposed to research cigarettes according to the Federal Trade Commission protocol (1 puff/min of 2-s duration and 35-ml volume) with fresh air being pumped in for the remaining time. Chamber concentration of smoke constituents was ⬃158 mg/m3 total suspended particulate. Nonsmoking control mice (CTL group, n ⫽ 10) were exposed to filtered air in an identical chamber according to the sample protocol described for CSE. Carboxyhemoglobin levels in blood samples after CSE confirm the correct exposition. Carboxyhemoglobin (COHb) formation is a wellrecognized effect of exposure to cigarette smoke. Increased levels of this form of hemoglobin indicate that the possibility of carbon monoxide poisoning should always be considered after exposure to cigarette smoke, and COHb levels should be routinely checked in such cases. On the other hand, relatively low levels of COHb and no clinical signs of carbon monoxide poisoning may suggest that, in our group, this kind of exposure did not play an important role. Therefore, immediately after removal of the animals from the smoke chamber, blood samples (⬃50 l) were collected in a heparinized tube by puncturing the retroorbital sinus in randomized samples of four mice per group once a month. COHb levels were measured spectrophotometrically with an IL 682 CO-Oximeter (Instrumentation Laboratory) to confirm a nontoxic and effective exposure. Target values for COHb in blood samples of AKR/J mice after CSE were within nontoxic 7–9% range, confirming the correct exposure to tobacco smoke (4). Importantly, on the basis of COHb blood measures, levels of smoke exposure were similar to those reported clinically (43). LGF treatment. LGF was purified from serum of 5-wk bile ductligated rats following a previously reported procedure (15). Purity,
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i.e., the absence of other growth factors and/or contaminants in the LGF preparation, was assessed according to standard criteria and activity checked before use. LGF preparations were lyophilized and stored at 4°C until used, at which time aliquots were dissolved in saline for intraperitoneal injection. The dose of LGF has been optimized in previous pilot experiments. Six months after CSE, when emphysema is once established, half of the smoker group animals were treated with four intraperitoneal injections (twice a week for 2 wk, n ⫽ 10) of 150 l of a solution containing 1.7 g LGF per mouse (CSE ⫹ LGF group) or saline solution (CSE group). Lung function: lung compliance assessment. The animals were initially anesthetized with isoflurane and subsequently with a sodium pentobarbital, 50 mg/kg body wt ip (Abbott Laboratories). Mice were tracheotomized, and an endotracheal tube (22GA) was inserted and tied to their trachea. As soon as the mouse was connected to the breathing equipment, the animal was paralyzed with 0.2 mg of pancuronium bromide and artificially ventilated with a ventilator, designed and manufactured in the Universidad Complutense de Madrid and optimized for small rodents (26, 36). Each animal was connected to the ventilator initially with a low tidal volume of 10 ml/kg. The normal breathing rate was set at a rate of 95 breaths/min, a moderate level of positive end-expiratory pressure (PEEP) of 4 cmH2O was set to prevent alveolar collapse (atelectasis), and a pressure of 12 cmH2O on PEEP was applied to ensure an adequate level of ventilation. To prevent preexisting atelectasis and to facilitate comparisons between curves obtained from different subjects, the volume data were normalized by total lung capacity (TLC), defined as the static lung volume at an inflation pressure of 25 cmH2O (23, 25). In this way, it was ensured that maximum lung capacity was reached without risking animal injury. For each animal, inspiration was controlled at a slow flow to 1 ml/s and free expiration. A new pressure-volume curve (P-V curve) cycle was started by pushing against air inside the lungs. This process was repeated for 30 s before the animal returned to normal mode of respiration. In this way, a total of seven to eight consecutive full P-V curves were acquired. Such a group of curves consists of one set of measurements. The changes in quasistatic lung compliance (CL, ml/cmH2O) after pressure-controlled ventilation were studied for each of the groups. Compliance was considered as the inflection point (maximum slope ⌬V/⌬P) in the expiratory limb of the curve, which brought closure pressure, at maximal alveolar derecruitment. The data were analyzed by fitting a sigmoidal function (22, 44) to the deflation limb of each P-V curve. Morphometric analysis in lung tissue sections: mean linear intercept quantification. After the 6-mo study period, the cardiopulmonary block was excised and fixed by filling it with 10% formaldehyde at a constant airway pressure of 25 cmH2O (TLC) for 24 h. Fixed lungs were immersed in alcohol-xylol baths of different concentrations; thereafter paraffin-embedded lung 5-m-thick sections were cut using a microtome HM325 (Microm), stained with hematoxylin-eosin (Sigma-Aldrich Química) according to standard protocols, and were observed with an Olympus BX40 microscope. Images were visualized by an adapted video camera (Leica DFC290, Leica Microsystems) with a resolution of 782 ⫻ 582 pixels (1 pixel ⫽ 0.146 m), attached to the microscope. Fields were quantified under ⫻40 objective and ⫻0.5 reducing video camera adapter. The degree of emphysema was assessed by measuring in the alveolus the mean linear intercept (LM, m) using standard image analysis software (Leica Qwin v3, Leica Microsystems). Each data point represents the mean value of at least 18 randomly selected fields from six slides obtained from each lung. In vivo detection of MMP activity by FMI. At 6 mo and 3 h after the last LGF injection, mice were injected via tail vein with MMPSense680, 2 nmol/150 l per mouse. MMPSense680 (PerkinElmer) is a protease-activatable fluorescent in vivo imaging agent that fluorochromes emit in the near-infrared (excitation: 680 nm; emission: 700 nm), allowing photons to penetrate for several centimeters in tissue. MMPSense680 is activated by key MMPs, including MMP-2, -3, -9, and -13, and is optically silent in its inactivated state and
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becomes highly fluorescent following the protease-mediated activation. The specificity of the fluorescent probe has already been proven by our group (33). Twenty-one hours after MMPSense680 injection, mice were anesthetized during the imaging acquisition with a mixture of 2% isoflurane and 2 l/min oxygen using an Inhalation Anesthesia System (Harvard Apparatus), and hair from the thorax was removed by shaving and chemical depilation to acquire fluorescence molecular imaging. Mice were imaged using IVIS-Lumina Imaging System (Xenogen). Gray scale-reflected images and fluorescence-colorized images were superimposed and analyzed using the Living Image 3.1 software (Xenogen). The Living Image software uses a spectral unmixing technique for reducing the effects of native tissue autofluorescence and calculates the respective contribution of each on each pixel in the image (46). RNA isolation and real-time PCR. Total RNA was isolated using Trizol reagent from frozen lung samples and reverse-transcribed to cDNA. For real-time PCR, we used TaqMan universal master mix and Taqman gene expression assays for the following genes: mmp9 (Mm00442991_m1) and mmp2 (Mm00439498_m1) from Applied Biosystems. The 2-⌬⌬CT method was applied to get the gene expression data using the Rn18s gene as an internal control to normalize the expression of the target genes mentioned above. Inflammatory cell infiltrate in bronchoalveolar lavage fluid. After the 6-mo study period, to perform bronchoalveolar lavage (BAL) fluid tests, animals were killed with an overdose of pentobarbital sodium (50 mg/kg body wt ip; Abbott Laboratories), and an endotracheal tube (22GA) was inserted to instill 1 ml of cold 0.9% NaCl and recover it by gentle manual aspiration. Cell viability was determined manually by using Trypan blue exclusion. For inflammatory cell measurement, the recovered BAL fluid was centrifuged at 600 g at 4°C for 5 min and decanted, and the BAL cell pellet was resuspended in 200 l of 0.9% NaCl; the total cell numbers were then determined by counting on a hemocytometer (cells ⫻ 103/ml). Determination of leukocyte population profile by flow cytometry. Immunostaining of BAL fluid and peripheral blood samples was performed using fluorochrome-conjugated monoclonal anti-mouse antibodies, CD3-FITC from BD Biosciences, Ly6B.2 (clone 7/4), Alexa Fluor647, and F4/80-Phycoerythrin from Serotec, directed at leukocyte surface markers to determine the T-lymphocytes, neutrophils, and alveolar macrophage/monocyte infiltration, respectively. The samples were stained, lysed, and washed before data acquisition on a dual-laser FACS Calibur flow cytometer running CELL Quest software (BD Biosciences) using an absolute counting protocol. List mode data were analyzed with Infinicyt software (Cytognos). Inflammatory cells were identified based on forward and side scatter characteristics (FSC and SSC): T-lymphocytes (CD3⫹ Ly6B.2⫺ F4/80⫺), neutrophils (CD3⫺ Ly6B.2hi F4/80⫺), and macrophages/monocytes (CD3⫺ Ly6B.2⫹ F4/80⫹). Evaluation of serum TNF-␣ level as systemic inflammatory response. After the 6-mo study period, blood samples of mice were collected, and serums were prepared. Quantitative determination of protein levels of the cytokine TNF-␣ was made using cytokine ELISA Kit (Thermo Fisher Scientific) according to manufacturer’s instruction. Briefly, 50 l of serum were loaded in triplicate in 96-well plates. The cytokine concentration was measured by optical densitometry at 450 nm in a SpectramaxPlus 384 microplate reader (Molecular Devices). The minimum detectable concentration was set to be 9 pg/ml (Thermo Fisher Scientific). Statistical analysis. Data are presented as means ⫾ SE. P values of ⬍0.05 were considered statistically significant. Variations in body weight over time were explored using t-tests and parametric tests. Mann-Whitney U-nonparametric test was performed for comparisons between groups of all the biological variables, followed by Monte Carlo’s exact methods within each set of comparisons, using the Statistical Package for the Social Science (SPSS) software.
RESULTS
LGF treatment improves the lung function impairment of emphysematous mice. Pulmonary emphysema induced by chronic CSE produced a significant increase in CL (ml/cmH2O) of CSE group (0.18 ml/cmH2O ⫾ 0.003) compared with CTL group (0.14 ml/cmH2O ⫾ 0.0014). LGF administration brought the functional variable closer to normal values, with statistically significant variations in CL of CSE ⫹ LGF group (0.12 ml/cmH2O ⫾ 0.008) vs. CSE group (Fig. 1). LGF treatment reduces the alveolar airspace enlargement in a previously established chronic CSE-induced emphysema murine model. Chronic CSE induced pulmonary emphysema with alveolar airspace enlargement. Morphometric analysis of LM (m) showed a significant increase in the CSE group (28.47 m ⫾ 0.58) compared with CTL group (25.40 m ⫾ 0.7). LGF treatment brought the LM morphometric variable closer to normal values, and significant reduction in the LM could be observed between CSE group and CSE ⫹ LGF group (25.54 m ⫾ 0.43) (Fig. 2A). Histological sections of lungs in CTL, after chronic CSE and after LGF treatment are shown in Fig. 2B. LGF treatment decreases chronic CSE-mediated lung tissue remodeling enzyme activity. In vivo detection by FMI of MMP activity in lung parenchyma revealed a significantly increased chronic CSE-associated MMP activity of the CSE group (1.85 ⫾ 0.05/U) compared with the control CTL group (1 ⫾ 0.12/U). This increment was significantly decreased in the CSE ⫹ LGF group (1.16 ⫾ 0.06/U) compared with the CSE group (Fig. 3A). When we analyzed fluorescent images, the units were the ratio between emission and excitation light and were referred to as relative fluorescence intensity (arbitrary units). Images of each representative mouse of the study groups are displayed in Fig. 3B. To confirm which MMP is responsible for the observed effect of LGF, gene expression data of the MMPs most involved in the development of CSE-induced emphysema are shown (Fig. 4). The MMP-2 mRNA level was significantly increased in the CSE group (1.43 ⫾ 0.03/U) compared with the CTL group (1 ⫾ 0.08/U). However, the increment of MMP-9
Fig. 1. Effect of liver growth factor (LGF) treatment on lung function of emphysematous mice. LGF treatment improves the lung function of emphysematous mice. Changes in lung compliance (CL, ml/cmH2O) for the groups of study are shown in the lungs of the cigarette smoke exposure (CSE) group (solid bar) and CSE ⫹ LGF group (shaded bar) in AKR/J mice compared with those in control (CTL) group (open bar) and CTL ⫹ LGF group (dashed bar). Data are expressed as means ⫾ SE (n ⫽ 8/group, *P ⬍ 0.05).
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00293.2013 • www.ajplung.org
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Fig. 2. Effect of LGF administration on lung morphometry after chronic CSE-induced emphysema. A: LGF treatment reduces the alveolar airspace enlargement in a previously established chronic CSE-induced emphysema murine model. Mean values of the mean linear intercept (LM), expressed in microns in the lungs of the CSE group (solid bar) and CSE ⫹ LGF group (shaded bar) in AKR/J mice compared with those in CTL group (open bar) and CTL ⫹ LGF group (dashed bar). Data are expressed as means ⫾ SE (n ⫽ 10/group, *P ⬍ 0.05). B: representative images (⫻10 magnification) corresponding to lung specimens. Note that the size of the alveoli is larger in the CSE than in the CTL and CTL ⫹ LGF mice, and this alveolar structure is practically reversed in the CSE ⫹ LGF group.
mRNA level was significantly decreased in the CSE ⫹ LGF treatment group (0.75 ⫾ 0.05/U) compared with the CSE group (1.23 ⫾ 0.05/U). LGF treatment decreases the inflammatory cell infiltrate in BAL fluid after chronic CSE. In the BAL fluid of CSE mice, there were significantly increased total cell numbers (1,027 ⫻ 103 ⫾ 73.04 cells/ml) compared with CTL mice (470 ⫻ 103 ⫾ 57.95 cells/ml). Furthermore, there was a significant decrease in the total number of cells of BAL in CSE ⫹ LGF mice (242 ⫻ 103 ⫾ 25.04 cells/ml) compared with CSE mice (Fig. 5). LGF treatment changes the leukocyte profile after chronic CSE. Leukocyte population profile in the BAL fluid (Fig. 6) and peripheral blood (Fig. 7) samples of CSE mice were quantified by flow cytometry: T-lymphocytes, neutrophils, and monocytes/macrophages. The number of each leukocyte subject was then expressed as a percentage of the total number of positive events from the respective control value (per unit). With respect to the leukocyte population in the BAL fluid (Fig. 6A), note the significant increase in alveolar macrophages in the CSE group (1.28 ⫾ 0.15/U) compared with the CTL group (1 ⫾ 0.08/U) and the increase in the number of neutrophils in the CSE ⫹ LGF group (2.71 ⫾ 0.18/U) following treatment with the growth factor, compared with the CTL group (1 ⫾ 0.04/U). Our results in the peripheral blood (Fig. 7A) show a significant increase in the percentage of T-lymphocytes (1.39 ⫾ 0.11%) of the CSE group with respect to the airexposed group (CTL, 1 ⫾ 0.06%) and a significant decrease in the percentage of monocytes (0.89 ⫾ 0.03%) in the peripheral blood of CSE group with respect to the air-exposed group (CTL, 1 ⫾ 0.05%). However, significant changes in the percentage of neutrophils were not appreciated. This significant increase of T-lymphocytes and decrease of monocytes in the peripheral blood were decreased (0.59 ⫾ 0.03%) and increased
(1.43 ⫾ 0.11%) respectively, after LGF treatment with respect to the corresponding CSE group. However, there were not significant differences in the percentage of neutrophils in the blood samples of mice treated with LGF after establishing emphysema. Leukocyte population was identified by flow cytometry analysis based on their characteristic properties shown in the FSC and SSC. Representative gating was set for CD3⫹ on T-lymphocytes, Ly6B.2hi on neutrophils, and F4/80⫹ on monocytes from BAL fluid and peripheral blood of mice (Figs. 6B and Fig. 7B). LGF treatment reduces the serum TNF-␣ level after chronic CSE. Levels of the cytokine TNF-␣ were significantly greater in the peripheral blood of the CSE mice (362 ⫾ 25.93 pg/ml) than CTL animals (267 ⫾ 9.73 pg/ml). LGF treatment brought TNF-␣ blood levels closer to normal values (315 ⫾ 20.25 pg/ml) but not significantly (Fig. 8). DISCUSSION
In the present study, it is demonstrated for the first time the LGF therapeutic effect on chronic CSE in a AKR/J mouse model of COPD, improving indicators and parameters of systemic inflammation, physiology, and morphology of the lungs. In this regard, the main findings in this study are that mice treated with LGF after chronic CSE (6 mo) exhibit the following modifications compared with control animals: 1) improved lung function impairment, 2) reduced alveolar airspace enlargement, 3) decreased chronic CSEmediated lung tissue remodeling enzyme activity, 4) reduced inflammatory cell infiltrate in BAL fluid, and 5) changed circulating leukocyte profile in peripheral blood, with decreasing T-lymphocyte population, tending to reduce neutrophil infiltration, and increasing monocyte population.
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00293.2013 • www.ajplung.org
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Fig. 3. Effect of LGF treatment on chronic CSEmediated matrix metalloproteinase (MMP) activity. A: LGF treatment decreases the chronic CSE-mediated lung tissue remodeling enzyme activity (arbitrary units from the control group, shown as per unit) in the lungs of the CSE group (solid bar) and CSE ⫹ LGF group (shaded bar) in AKR/J mice compared with those in CTL group (open bar). Data are expressed as means ⫾ SE (n ⫽ 4/group, *P ⬍ 0.05). B: lung imaging in vivo of 1 representative mouse of each group. Male mice were anesthetized and imaged using an IVIS Imaging System after 6 mo of CSE. The entire efficiency signal from the thorax was measured with Living Image Software by using spectral unmixing technique.
Considering these data, we suggest that LGF could be a relevant anti-inflammatory and lung structure and function regenerative molecule, particularly in situations of lung injury induced by CSE. COPD is associated with systemic effects, including reduced body weight gain and altered circulating leukocytes and plasma TNF-␣ levels. CSE mice gained body weight, although to a lesser extent compared with CTL animals, up to the third month of exposure. Although not specifically quantified, food intake between CSE and CTL rodents was similar, even after 3 mo of study (animals in both groups were always fed ad libitum, receiving an identical amount of food/day per animal in the cages). The present findings are in line with previous
investigations, where animals chronically exposed to cigarette smoke also underwent a significant reduction in body weight gain (2, 20). The long-term intraperitoneal administration of LGF doses (1.7 g/animal) in AKR/J mice was well tolerated, with no toxicity in terms of body weight gain. In summary, AKR/J mice chronic exposure to cigarette smoke induces lung emphysema concomitantly with greater modifications on body weight gain, total cell count in BAL fluid, MMP activity in living mice, physiological and morphological lung parameters, serum TNF-␣ levels, and change in the profile of circulating leukocytes. Treatment with LGF seems to reverse all these parameters studied, except the loss of body weight and TNF-␣ level, although the latter if it tends to decrease.
Fig. 4. Analysis of MMP gene expression by real-time PCR. The MMP-2 and MMP-9 mRNA levels relative to 18S rRNA are shown. MMP-2 increases in CSE group (solid bar), and LGF treatment decreases the MMP-9 expression in the CSE ⫹ LGF group (shaded bar) compared with CTL group (open bar). Results are expressed as means ⫾ SE (n ⫽ 6/group, *P ⬍ 0.05) in % relative to nonexposed and nontreated mice that were arbitrarily assigned a value of 1%.
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00293.2013 • www.ajplung.org
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Fig. 5. Effect of LGF treatment on inflammatory cell infiltrate in the bronchoalveolar lavage (BAL) fluid of emphysematous mice. LGF treatment (shaded bar) decreases the inflammatory cell infiltrate in the BAL fluid after chronic CSE (solid bar) compared with CTL group (open bar). The data represent means ⫾ SE of the total cell number (⫻103/ml) in the BAL fluid of different groups (n ⫽ 8/group, *P ⬍ 0.05).
In the current investigation, AKR/J mice exposed to cigarette smoke for 6 mo exhibited a significant increase in the CL (28%) and in the LM, alveolar airspace enlargement (12%), suggesting the development of emphysema in these animals compared with nonexposed control animals. These findings are in agreement with other previous studies (20). Both of these
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parameters were reduced with the LGF treatment by 50% and 11%, respectively. The presence of inflammation after chronic CSE in AKR/J mice was confirmed by increased inflammatory cell infiltrate in BAL fluid and by increased lung tissue remodeling enzyme activity measured in living mice. These findings are in agreement with other previous studies (47), but the novelty is the technique used to measure lung protease activity in vivo. Enzyme-based activatable probes for fluorescence-mediated imaging has proven valuable for the detection of tumor-associated lysosomal protease activity in an animal model (5, 45), and we have recently demonstrated that MMP-activatable probe can be used to detect MMP activity in the lung tissue after acute CSE (33). With the help of an activatable sensor that reports activity, we have demonstrated in the present study that MMP activity, visualized by fluorescence molecular imaging in the lung of living mice, is significantly decreased in LGF-treated mice after smoking-induced emphysema. The data presented herein serve to further evince molecular imaging as a technology highly appropriate for chronic lung inflammatory research. LGF treatment appears to reverse gene expression of MMP-9 in the lung tissue but not MMP-2. MMP-9 is produced mainly by inflammatory cells and could be rather linked to inflammatory process-induced tissue remodeling (10, 19, 40). However, MMP-2 is synthesized by structural cells and is more associated with the impaired tissue remodeling, leading to pathological collagen deposition and interstitial fibrosis. We consider that decreasing MMP-9 participates in early changes, as a consequence of the anti-inflammatory effect of LGF and MMP2 could possibly decrease later, while the process continues. We notice that the reversibility changes were studied only 15 days after LGF administration.
Fig. 6. Effect of LGF treatment on the leukocyte population profile in BAL fluid of CSE mice. A: LGF treatment changes the leukocyte profile in BAL fluid after chronic CSE. Shown are the percentages of T-lymphocytes, neutrophils, and alveolar macrophages in the BAL fluid of the CSE group (solid bar) and CSE ⫹ LGF group (shaded bar) compared with those in CTL group (open bar). Data are expressed as means ⫾ SE (percentage of cells, per unit from the control group); *P ⬍ 0.05; n ⫽ 6/group. B: representative gating was set for CD3⫹ on T-lymphocytes (red), Ly6B.2hi on neutrophils (yellow), and F4/80⫹ on alveolar macrophages (blue) from BAL fluid of mice. FSC, forward scatter plot; SSC, side scatter plot. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00293.2013 • www.ajplung.org
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Fig. 7. Effect of LGF treatment on the circulating leukocyte population profile in peripheral blood of CSE mice. A: LGF treatment changes the leukocyte profile in peripheral blood after chronic CSE. Shown are the percentages of T-lymphocytes, neutrophils, and monocytes in the peripheral blood samples of the CSE group (solid bar) and CSE ⫹ LGF group (shaded bar) compared with those in CTL group (open bar). Data are expressed as means ⫾ SE (percentage of cells, per unit from the control group); *P ⬍ 0.05; n ⫽ 8/group. B: representative gating was set for CD3⫹ on T-lymphocytes (red), Ly6B.2hi on neutrophils (yellow), and F4/80⫹ on monocytes (blue) from peripheral blood of mice.
Systemic inflammation in COPD is also characterized by cytokines systemically implicated in inflammatory cell recruitment and airway remodeling. Our data suggest that animals with chronic airflow limitation had significantly raised levels of various systemic inflammatory markers like circulating leukocytes (specifically T-cells and neutrophils) and serum TNF-␣ level, indicating that persistent systemic inflammation is present in AKR/J. LGF treatment decreases T-lymphocyte population in peripheral blood, what seems to be in agreement with a recently published article that shows that T-cell depletion protects against alveolar destruction attributable to chronic CSE in mice (37). It is possible in some cases that the inflammatory process may “spill” over into the systemic circulation, promoting a generalized inflammatory reaction. Our findings are consistent with systemic inflammation present in some patients with COPD (1) associated with an increased influx of T-cells into the airway (6, 28, 31, 37). This finding may explain, at least in part, the high prevalence of systemic complications, such as cachexia, osteoporosis, and cardiovascular diseases among patients with COPD. Future studies are needed to determine whether attenuation of the systemic inflammatory process can modify the risk of these complications in COPD. In view of the results obtained in this study, what seems clear is that LGF treatment promotes tissue regeneration, probably by promoting cell proliferation (7, 18, 39, 42) of the lung parenchyma. Maybe the LGF stimulates cellular antioxidant response (8, 9) or decreases cellular apoptotic processes (13). However, further studies are necessary to get to know the mechanism by which it acts. In summary, there is now a large
body of evidence to indicate that LGF treatment reverses emphysema previously established in a chronic CSE mouse model, normalizing the physiological and morphological data and levels of various systemic inflammatory markers, which may have important pathophysiological and therapeutic implications for subjects with stable COPD.
Fig. 8. Effect of LGF treatment on serum TNF-␣ level after chronic CSE. LGF treatment decreases the serum TNF-␣ level after chronic CSE. Mean values of the TNF-␣ expressed as pg/ml in the blood samples of the CSE group (solid bar) and CSE ⫹ LGF group (shaded bar) in AKR/J mice compared with those in CTL group (open bar). Levels of the cytokine TNF-␣ were significantly greater in the blood of CSE mice compared with CTL animals. Data are expressed as means ⫾ SE (n ⫽ 8/group, **P ⬍ 0.05).
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00293.2013 • www.ajplung.org
LGF REVERSES EMPHYSEMA MOUSE MODEL ACKNOWLEDGMENTS We thank D. Angelos Kyriazis, Institute of Biofunctional Studies, Complutense University of Madrid - Spain, for his help with the P-V curve acquisition with the ventilator. GRANTS This study was supported by the Spanish Society of Pulmonology and Thoracic Surgery (SEPAR-139). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: S.P.-R. and G.P.-B. conception and design of research; S.P.-R., L.d.P.-N., Á.G.-M., and R.T.-E. analyzed data; S.P.-R., L.d.P.N., Á.G.-M., R.T.-E., and J.D.-G. interpreted results of experiments; S.P.-R., L.d.P.-N., and G.P.-B. drafted manuscript; S.P.-R., J.D.-G., and N.G.-M. edited and revised manuscript; S.P.-R., N.G.-M., and G.P.-B. approved final version of manuscript; L.d.P.-N., Á.G.-M., and R.T.-E. performed experiments; L.d.P.-N., Á.G.-M., and R.T.-E. prepared figures. REFERENCES 1. Agusti A, Edwards LD, Rennard SI, MacNee W, Tal-Singer R, Miller BE, Vestbo J, Lomas DA, Calverley PM, Wouters E, Crim C, Yates JC, Silverman EK, Coxson HO, Bakke P, Mayer RJ, Celli B. Persistent systemic inflammation is associated with poor clinical outcomes in COPD: a novel phenotype. PLoS One 7: e37483, 2012. 2. Ardite E, Peinado VI, Rabinovich RA, Fernandez-Checa JC, Roca J, Barbera JA. Systemic effects of cigarette smoke exposure in the guinea pig. Respir Med 100: 1186 –1194, 2006. 3. Barnes PJ, Hansel TT. Prospects for new drugs for chronic obstructive pulmonary disease. Lancet 364: 985–996, 2004. 4. Barreiro E, del Puerto-Nevado L, Puig-Vilanova E, Perez-Rial S, Sanchez F, Martinez-Galan L, Rivera S, Gea J, Gonzalez-Mangado N, Peces-Barba G. Cigarette smoke-induced oxidative stress in skeletal muscles of mice. Respir Physiol Neurobiol 182: 9 –17, 2012. 5. Bremer C, Tung CH, Weissleder R. In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat Med 7: 743–748, 2001. 6. Brozyna S, Ahern J, Hodge G, Nairn J, Holmes M, Reynolds PN, Hodge S. Chemotactic mediators of Th1 T-cell trafficking in smokers and COPD patients. COPD 6: 4 –16, 2009. 7. Conde MV, Gonzalez MC, Quintana-Villamandos B, Abderrahim F, Briones AM, Condezo-Hoyos L, Regadera J, Susin C, Gomez de Diego JJ, Delgado-Baeza E, Diaz-Gil JJ, Arribas SM. Liver growth factor treatment restores cell-extracellular matrix balance in resistance arteries and improves left ventricular hypertrophy in SHR. Am J Physiol Heart Circ Physiol 301: H1153–H1165, 2011. 8. Condezo-Hoyos L, Abderrahim F, Conde MV, Susin C, Diaz-Gil JJ, Gonzalez MC, Arribas SM. Antioxidant activity of liver growth factor, a bilirubin covalently bound to albumin. Free Radic Biol Med 46: 656 –662, 2009. 9. Condezo-Hoyos L, Arribas SM, Abderrahim F, Somoza B, Gil-Ortega M, Diaz-Gil JJ, Conde MV, Susin C, Gonzalez MC. Liver growth factor treatment reverses vascular and plasmatic oxidative stress in spontaneously hypertensive rats. J Hypertens 30: 1185–1194, 2012. 10. Chakrabarti S, Patel KD. Matrix metalloproteinase-2 (MMP-2) and MMP-9 in pulmonary pathology. Exp Lung Res 31: 599 –621, 2005. 11. Churg A, Wang RD, Tai H, Wang X, Xie C, Dai J, Shapiro SD, Wright JL. Macrophage metalloelastase mediates acute cigarette smokeinduced inflammation via tumor necrosis factor-alpha release. Am J Respir Crit Care Med 167: 1083–1089, 2003. 12. Diaz-Gil JJ, Escartin P, Garcia-Canero R, Trilla C, Veloso JJ, Sanchez G, Moreno-Caparros A, Enrique de Salamanca C, Lozano R, Gavilanes JG, García-Segura JM. Purification of a liver DNA-synthesis promoter from plasma of partially hepatectomized rats. Biochem J 235: 49 –55, 1986. 13. Diaz-Gil JJ, Garcia-Monzon C, Rua C, Martin-Sanz P, Cereceda RM, Miquilena-Colina ME, Machin C, Fernandez-Martinez A, GarciaCanero R. Liver growth factor antifibrotic activity in vivo is associated with a decrease in activation of hepatic stellate cells. Histol Histopathol 24: 473–479, 2009.
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Liver growth factor treatment reverses emphysema previously established in a cigarette smoke exposure mouse model Sandra Pérez-Rial, Laura del Puerto-Nevado, Álvaro Girón-Martínez, Raúl Terrón-Expósito, Juan J. Díaz-Gil, Nicolás González-Mangado, and Germán Peces-Barba Respiratory Research Group, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz-CIBERES (IIS-FJD-CIBERES), Madrid, Spain Submitted 15 October 2013; accepted in final form 28 August 2014
Pérez-Rial S, del Puerto-Nevado L, Girón-Martínez Á, TerrónExpósito R, Díaz-Gil JJ, González-Mangado N, Peces-Barba G. Liver growth factor treatment reverses emphysema previously established in a cigarette smoke exposure mouse model. Am J Physiol Lung Cell Mol Physiol 307: L718 –L726, 2014. First published August 29, 2014; doi:10.1152/ajplung.00293.2013.—Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease largely associated with cigarette smoke exposure (CSE) and characterized by pulmonary and extrapulmonary manifestations, including systemic inflammation. Liver growth factor (LGF) is an albumin-bilirubin complex with demonstrated antifibrotic, antioxidant, and antihypertensive actions even at extrahepatic sites. We aimed to determine whether short LGF treatment (1.7 g/mouse ip; 2 times, 2 wk), once the lung damage was established through the chronic CSE, contributes to improvement of the regeneration of damaged lung tissue, reducing systemic inflammation. We studied AKR/J mice, divided into three groups: control (air-exposed), CSE (chronic CSE), and CSE ⫹ LGF (LGF-treated CSE mice). We assessed pulmonary function, morphometric data, and levels of various systemic inflammatory markers to test the LGF regenerative capacity in this system. Our results revealed that the lungs of the CSE animals showed pulmonary emphysema and inflammation, characterized by increased lung compliance, enlargement of alveolar airspaces, systemic inflammation (circulating leukocytes and serum TNF-␣ level), and in vivo lung matrix metalloproteinase activity. LGF treatment was able to reverse all these parameters, decreasing total cell count in bronchoalveolar lavage fluid and T-lymphocyte infiltration in peripheral blood observed in emphysematous mice and reversing the decrease in monocytes observed in chronic CSE mice, and tends to reduce the neutrophil population and serum TNF-␣ level. In conclusion, LGF treatment normalizes the physiological and morphological parameters and levels of various systemic inflammatory biomarkers in a chronic CSE AKR/J model, which may have important pathophysiological and therapeutic implications for subjects with stable COPD. chronic cigarette smoke exposure; emphysema; liver growth factor; mice model; therapy CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) is a major cause of illness and death throughout the world, with the main risk factor as cigarette smoke, and increasing evidence suggests that COPD is a disease characterized by both local and systemic inflammation. Systemic inflammation in COPD is characterized by cytokines implicated in inflammatory cell recruitment and airway remodeling. Reduced lung function is associated with increased levels of various systemic inflammatory markers (16). Emphysema, a component of COPD, is
Address for reprint requests and other correspondence: Sandra Pérez-Rial, Respiratory Research Group, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz-CIBERES (IIS-FJD-CIBERES), Avenida Reyes Católicos, 2, 28040, Madrid, SPAIN (e-mail: [email protected]). L718
characterized by an inflammatory cell infiltrate and by the presence of proteinases in excess within the alveolar space, which leads to destruction of alveolar walls and permanent enlargement of the peripheral airspaces of the lung, and no treatment is currently available to reverse the lung destruction and induce proper healing. The target of newer treatments being developed is primarily directed toward stopping the worsening of the disease, either by inhibiting inflammation and the overproduction of mucus, or by employing antioxidant and antifibrotic agents (3). Liver growth factor (LGF) was identified as an albuminbilirubin complex with mitogenic properties in rat liver (12). It has antifibrotic, antioxidant, and antihypertensive properties, and its regenerative effects have been described in several rodent models, including hepatic damage (13, 14), cardiovascular diseases such as hypertension and atherosclerosis lesions (7, 8, 42), Parkinson’s disease (17, 38, 39), and testicular degeneration (32). On the other hand, data published in lung fibrosis by our group show the antifibrotic effects of LGF in rats induced with ClCd2 (30). Considering the regenerative activity in different systems, LGF could be suggested as a promising choice for the stimulation of processes involved in tissue repair in previously damaged lungs in a known model of COPD in mice. Therefore, our goal is to test the effects of LGF administration in a mouse model of emphysema induced by chronic cigarette smoke exposure (CSE). In several investigations, it has been shown that the development of emphysema in mice chronically exposed to cigarette smoke is highly dependent on the mouse strain and dose (20, 47). In this regard, the AKR/J strain was shown to be extremely susceptible to the development of emphysema, as measured by increases in the airspace enlargement and the elastic properties of the lung, after chronic exposure to cigarette smoke (20). Likewise, the cellular and molecular inflammatory response was also shown to be more prominent in the lungs of the AKR/J mice chronically exposed to cigarette smoke than in the other mouse strains investigated in the study in question (20). Fluorescence molecular imaging (FMI) is the method of choice for functional, real-time, in vivo monitoring of matrix metalloproteinase (MMP) activity because it provides an “easy” approach for measuring enzymatic activities (21, 29, 45). Here we use an injectable pan-MMP-targeted optical sensor that specifically and semiquantitatively resolves MMP-2, -3, -9, and -13 activities that initiate and propagate inflammatory responses (11, 19, 27, 33) in the lungs of mice with experimental chronic CSE-induced emphysema. Several studies have suggested that MMPs are the critical mediators of CSE-induced emphysema (11, 24, 41).
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LGF REVERSES EMPHYSEMA MOUSE MODEL
The purpose of this study was to demonstrate the therapeutic effect of LGF treatment improving the inflammatory status, visualized and quantified by in vivo FMI in the lung of a chronic inflammation after CSE-induced emphysema in AKR/J mouse model, including improvement of lung function and morphometry parameters. MATERIALS AND METHODS
Animals. Thirty adult, 8-wk-old male AKR/J mice, 25–27 g body wt (The Jackson Laboratory, Bar Harbor, ME), were divided in three groups (n ⫽ 10/group): control (CTL), CSE, and CSE with LGF treatment (CSE ⫹ LGF). Animals were housed in the Inhalation Core Facility at IIS-Fundación Jiménez Díaz for 1 wk of acclimatization before the experimental procedures. After daily exposure, mice were returned to their cages, where a diet of alfalfa-free rodent food (Harlan Teklad) and water were provided ad libitum. Protocols were approved by the local Ethical Animal Research Committee at IIS-Fundación Jiménez Díaz. In all cases, the legislation regarding animal treatment, protection, and handling was followed (RD 53/2013). All animals were weighed on a monthly basis during the 6-mo study period. AKR/J mice after chronic CSE for 6 mo exhibited a significantly lesser increase in their body weights compared with the corresponding control animals. Indeed, at 6 mo, mice exposed to tobacco smoke presented a minor increase in their body weight gain, (34% of baseline weight) compared with the CTL group (65% of baseline weight) (4). No significant differences were appreciated in the body weight gain in the group of mice treated with LGF. Chronic CSE-induced emphysema model. Chronic CSE was performed in 20 AKR/J mice (CSE group) with a daily exposition, 5 days a week, during a continuous period of 6 mo. The smoke from two consecutive (with 5-min breaks between them) standard research nonfiltered cigarettes was used (2R1, Cigarette Laboratory at the Tobacco and Health Research Institute, University of Kentucky, Lexington, KY), containing 11.7 mg total particulate matter per cubic meter of air, 9.7 mg tar, and 0.85 mg nicotine per cigarette. Mainstream CSE was generated by an exposure system and was drawn into the chambers via peristaltic pump, following previously published methodologies (4, 33, 35). The whole body of the animals was exposed to research cigarettes according to the Federal Trade Commission protocol (1 puff/min of 2-s duration and 35-ml volume) with fresh air being pumped in for the remaining time. Chamber concentration of smoke constituents was ⬃158 mg/m3 total suspended particulate. Nonsmoking control mice (CTL group, n ⫽ 10) were exposed to filtered air in an identical chamber according to the sample protocol described for CSE. Carboxyhemoglobin levels in blood samples after CSE confirm the correct exposition. Carboxyhemoglobin (COHb) formation is a wellrecognized effect of exposure to cigarette smoke. Increased levels of this form of hemoglobin indicate that the possibility of carbon monoxide poisoning should always be considered after exposure to cigarette smoke, and COHb levels should be routinely checked in such cases. On the other hand, relatively low levels of COHb and no clinical signs of carbon monoxide poisoning may suggest that, in our group, this kind of exposure did not play an important role. Therefore, immediately after removal of the animals from the smoke chamber, blood samples (⬃50 l) were collected in a heparinized tube by puncturing the retroorbital sinus in randomized samples of four mice per group once a month. COHb levels were measured spectrophotometrically with an IL 682 CO-Oximeter (Instrumentation Laboratory) to confirm a nontoxic and effective exposure. Target values for COHb in blood samples of AKR/J mice after CSE were within nontoxic 7–9% range, confirming the correct exposure to tobacco smoke (4). Importantly, on the basis of COHb blood measures, levels of smoke exposure were similar to those reported clinically (43). LGF treatment. LGF was purified from serum of 5-wk bile ductligated rats following a previously reported procedure (15). Purity,
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i.e., the absence of other growth factors and/or contaminants in the LGF preparation, was assessed according to standard criteria and activity checked before use. LGF preparations were lyophilized and stored at 4°C until used, at which time aliquots were dissolved in saline for intraperitoneal injection. The dose of LGF has been optimized in previous pilot experiments. Six months after CSE, when emphysema is once established, half of the smoker group animals were treated with four intraperitoneal injections (twice a week for 2 wk, n ⫽ 10) of 150 l of a solution containing 1.7 g LGF per mouse (CSE ⫹ LGF group) or saline solution (CSE group). Lung function: lung compliance assessment. The animals were initially anesthetized with isoflurane and subsequently with a sodium pentobarbital, 50 mg/kg body wt ip (Abbott Laboratories). Mice were tracheotomized, and an endotracheal tube (22GA) was inserted and tied to their trachea. As soon as the mouse was connected to the breathing equipment, the animal was paralyzed with 0.2 mg of pancuronium bromide and artificially ventilated with a ventilator, designed and manufactured in the Universidad Complutense de Madrid and optimized for small rodents (26, 36). Each animal was connected to the ventilator initially with a low tidal volume of 10 ml/kg. The normal breathing rate was set at a rate of 95 breaths/min, a moderate level of positive end-expiratory pressure (PEEP) of 4 cmH2O was set to prevent alveolar collapse (atelectasis), and a pressure of 12 cmH2O on PEEP was applied to ensure an adequate level of ventilation. To prevent preexisting atelectasis and to facilitate comparisons between curves obtained from different subjects, the volume data were normalized by total lung capacity (TLC), defined as the static lung volume at an inflation pressure of 25 cmH2O (23, 25). In this way, it was ensured that maximum lung capacity was reached without risking animal injury. For each animal, inspiration was controlled at a slow flow to 1 ml/s and free expiration. A new pressure-volume curve (P-V curve) cycle was started by pushing against air inside the lungs. This process was repeated for 30 s before the animal returned to normal mode of respiration. In this way, a total of seven to eight consecutive full P-V curves were acquired. Such a group of curves consists of one set of measurements. The changes in quasistatic lung compliance (CL, ml/cmH2O) after pressure-controlled ventilation were studied for each of the groups. Compliance was considered as the inflection point (maximum slope ⌬V/⌬P) in the expiratory limb of the curve, which brought closure pressure, at maximal alveolar derecruitment. The data were analyzed by fitting a sigmoidal function (22, 44) to the deflation limb of each P-V curve. Morphometric analysis in lung tissue sections: mean linear intercept quantification. After the 6-mo study period, the cardiopulmonary block was excised and fixed by filling it with 10% formaldehyde at a constant airway pressure of 25 cmH2O (TLC) for 24 h. Fixed lungs were immersed in alcohol-xylol baths of different concentrations; thereafter paraffin-embedded lung 5-m-thick sections were cut using a microtome HM325 (Microm), stained with hematoxylin-eosin (Sigma-Aldrich Química) according to standard protocols, and were observed with an Olympus BX40 microscope. Images were visualized by an adapted video camera (Leica DFC290, Leica Microsystems) with a resolution of 782 ⫻ 582 pixels (1 pixel ⫽ 0.146 m), attached to the microscope. Fields were quantified under ⫻40 objective and ⫻0.5 reducing video camera adapter. The degree of emphysema was assessed by measuring in the alveolus the mean linear intercept (LM, m) using standard image analysis software (Leica Qwin v3, Leica Microsystems). Each data point represents the mean value of at least 18 randomly selected fields from six slides obtained from each lung. In vivo detection of MMP activity by FMI. At 6 mo and 3 h after the last LGF injection, mice were injected via tail vein with MMPSense680, 2 nmol/150 l per mouse. MMPSense680 (PerkinElmer) is a protease-activatable fluorescent in vivo imaging agent that fluorochromes emit in the near-infrared (excitation: 680 nm; emission: 700 nm), allowing photons to penetrate for several centimeters in tissue. MMPSense680 is activated by key MMPs, including MMP-2, -3, -9, and -13, and is optically silent in its inactivated state and
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becomes highly fluorescent following the protease-mediated activation. The specificity of the fluorescent probe has already been proven by our group (33). Twenty-one hours after MMPSense680 injection, mice were anesthetized during the imaging acquisition with a mixture of 2% isoflurane and 2 l/min oxygen using an Inhalation Anesthesia System (Harvard Apparatus), and hair from the thorax was removed by shaving and chemical depilation to acquire fluorescence molecular imaging. Mice were imaged using IVIS-Lumina Imaging System (Xenogen). Gray scale-reflected images and fluorescence-colorized images were superimposed and analyzed using the Living Image 3.1 software (Xenogen). The Living Image software uses a spectral unmixing technique for reducing the effects of native tissue autofluorescence and calculates the respective contribution of each on each pixel in the image (46). RNA isolation and real-time PCR. Total RNA was isolated using Trizol reagent from frozen lung samples and reverse-transcribed to cDNA. For real-time PCR, we used TaqMan universal master mix and Taqman gene expression assays for the following genes: mmp9 (Mm00442991_m1) and mmp2 (Mm00439498_m1) from Applied Biosystems. The 2-⌬⌬CT method was applied to get the gene expression data using the Rn18s gene as an internal control to normalize the expression of the target genes mentioned above. Inflammatory cell infiltrate in bronchoalveolar lavage fluid. After the 6-mo study period, to perform bronchoalveolar lavage (BAL) fluid tests, animals were killed with an overdose of pentobarbital sodium (50 mg/kg body wt ip; Abbott Laboratories), and an endotracheal tube (22GA) was inserted to instill 1 ml of cold 0.9% NaCl and recover it by gentle manual aspiration. Cell viability was determined manually by using Trypan blue exclusion. For inflammatory cell measurement, the recovered BAL fluid was centrifuged at 600 g at 4°C for 5 min and decanted, and the BAL cell pellet was resuspended in 200 l of 0.9% NaCl; the total cell numbers were then determined by counting on a hemocytometer (cells ⫻ 103/ml). Determination of leukocyte population profile by flow cytometry. Immunostaining of BAL fluid and peripheral blood samples was performed using fluorochrome-conjugated monoclonal anti-mouse antibodies, CD3-FITC from BD Biosciences, Ly6B.2 (clone 7/4), Alexa Fluor647, and F4/80-Phycoerythrin from Serotec, directed at leukocyte surface markers to determine the T-lymphocytes, neutrophils, and alveolar macrophage/monocyte infiltration, respectively. The samples were stained, lysed, and washed before data acquisition on a dual-laser FACS Calibur flow cytometer running CELL Quest software (BD Biosciences) using an absolute counting protocol. List mode data were analyzed with Infinicyt software (Cytognos). Inflammatory cells were identified based on forward and side scatter characteristics (FSC and SSC): T-lymphocytes (CD3⫹ Ly6B.2⫺ F4/80⫺), neutrophils (CD3⫺ Ly6B.2hi F4/80⫺), and macrophages/monocytes (CD3⫺ Ly6B.2⫹ F4/80⫹). Evaluation of serum TNF-␣ level as systemic inflammatory response. After the 6-mo study period, blood samples of mice were collected, and serums were prepared. Quantitative determination of protein levels of the cytokine TNF-␣ was made using cytokine ELISA Kit (Thermo Fisher Scientific) according to manufacturer’s instruction. Briefly, 50 l of serum were loaded in triplicate in 96-well plates. The cytokine concentration was measured by optical densitometry at 450 nm in a SpectramaxPlus 384 microplate reader (Molecular Devices). The minimum detectable concentration was set to be 9 pg/ml (Thermo Fisher Scientific). Statistical analysis. Data are presented as means ⫾ SE. P values of ⬍0.05 were considered statistically significant. Variations in body weight over time were explored using t-tests and parametric tests. Mann-Whitney U-nonparametric test was performed for comparisons between groups of all the biological variables, followed by Monte Carlo’s exact methods within each set of comparisons, using the Statistical Package for the Social Science (SPSS) software.
RESULTS
LGF treatment improves the lung function impairment of emphysematous mice. Pulmonary emphysema induced by chronic CSE produced a significant increase in CL (ml/cmH2O) of CSE group (0.18 ml/cmH2O ⫾ 0.003) compared with CTL group (0.14 ml/cmH2O ⫾ 0.0014). LGF administration brought the functional variable closer to normal values, with statistically significant variations in CL of CSE ⫹ LGF group (0.12 ml/cmH2O ⫾ 0.008) vs. CSE group (Fig. 1). LGF treatment reduces the alveolar airspace enlargement in a previously established chronic CSE-induced emphysema murine model. Chronic CSE induced pulmonary emphysema with alveolar airspace enlargement. Morphometric analysis of LM (m) showed a significant increase in the CSE group (28.47 m ⫾ 0.58) compared with CTL group (25.40 m ⫾ 0.7). LGF treatment brought the LM morphometric variable closer to normal values, and significant reduction in the LM could be observed between CSE group and CSE ⫹ LGF group (25.54 m ⫾ 0.43) (Fig. 2A). Histological sections of lungs in CTL, after chronic CSE and after LGF treatment are shown in Fig. 2B. LGF treatment decreases chronic CSE-mediated lung tissue remodeling enzyme activity. In vivo detection by FMI of MMP activity in lung parenchyma revealed a significantly increased chronic CSE-associated MMP activity of the CSE group (1.85 ⫾ 0.05/U) compared with the control CTL group (1 ⫾ 0.12/U). This increment was significantly decreased in the CSE ⫹ LGF group (1.16 ⫾ 0.06/U) compared with the CSE group (Fig. 3A). When we analyzed fluorescent images, the units were the ratio between emission and excitation light and were referred to as relative fluorescence intensity (arbitrary units). Images of each representative mouse of the study groups are displayed in Fig. 3B. To confirm which MMP is responsible for the observed effect of LGF, gene expression data of the MMPs most involved in the development of CSE-induced emphysema are shown (Fig. 4). The MMP-2 mRNA level was significantly increased in the CSE group (1.43 ⫾ 0.03/U) compared with the CTL group (1 ⫾ 0.08/U). However, the increment of MMP-9
Fig. 1. Effect of liver growth factor (LGF) treatment on lung function of emphysematous mice. LGF treatment improves the lung function of emphysematous mice. Changes in lung compliance (CL, ml/cmH2O) for the groups of study are shown in the lungs of the cigarette smoke exposure (CSE) group (solid bar) and CSE ⫹ LGF group (shaded bar) in AKR/J mice compared with those in control (CTL) group (open bar) and CTL ⫹ LGF group (dashed bar). Data are expressed as means ⫾ SE (n ⫽ 8/group, *P ⬍ 0.05).
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00293.2013 • www.ajplung.org
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Fig. 2. Effect of LGF administration on lung morphometry after chronic CSE-induced emphysema. A: LGF treatment reduces the alveolar airspace enlargement in a previously established chronic CSE-induced emphysema murine model. Mean values of the mean linear intercept (LM), expressed in microns in the lungs of the CSE group (solid bar) and CSE ⫹ LGF group (shaded bar) in AKR/J mice compared with those in CTL group (open bar) and CTL ⫹ LGF group (dashed bar). Data are expressed as means ⫾ SE (n ⫽ 10/group, *P ⬍ 0.05). B: representative images (⫻10 magnification) corresponding to lung specimens. Note that the size of the alveoli is larger in the CSE than in the CTL and CTL ⫹ LGF mice, and this alveolar structure is practically reversed in the CSE ⫹ LGF group.
mRNA level was significantly decreased in the CSE ⫹ LGF treatment group (0.75 ⫾ 0.05/U) compared with the CSE group (1.23 ⫾ 0.05/U). LGF treatment decreases the inflammatory cell infiltrate in BAL fluid after chronic CSE. In the BAL fluid of CSE mice, there were significantly increased total cell numbers (1,027 ⫻ 103 ⫾ 73.04 cells/ml) compared with CTL mice (470 ⫻ 103 ⫾ 57.95 cells/ml). Furthermore, there was a significant decrease in the total number of cells of BAL in CSE ⫹ LGF mice (242 ⫻ 103 ⫾ 25.04 cells/ml) compared with CSE mice (Fig. 5). LGF treatment changes the leukocyte profile after chronic CSE. Leukocyte population profile in the BAL fluid (Fig. 6) and peripheral blood (Fig. 7) samples of CSE mice were quantified by flow cytometry: T-lymphocytes, neutrophils, and monocytes/macrophages. The number of each leukocyte subject was then expressed as a percentage of the total number of positive events from the respective control value (per unit). With respect to the leukocyte population in the BAL fluid (Fig. 6A), note the significant increase in alveolar macrophages in the CSE group (1.28 ⫾ 0.15/U) compared with the CTL group (1 ⫾ 0.08/U) and the increase in the number of neutrophils in the CSE ⫹ LGF group (2.71 ⫾ 0.18/U) following treatment with the growth factor, compared with the CTL group (1 ⫾ 0.04/U). Our results in the peripheral blood (Fig. 7A) show a significant increase in the percentage of T-lymphocytes (1.39 ⫾ 0.11%) of the CSE group with respect to the airexposed group (CTL, 1 ⫾ 0.06%) and a significant decrease in the percentage of monocytes (0.89 ⫾ 0.03%) in the peripheral blood of CSE group with respect to the air-exposed group (CTL, 1 ⫾ 0.05%). However, significant changes in the percentage of neutrophils were not appreciated. This significant increase of T-lymphocytes and decrease of monocytes in the peripheral blood were decreased (0.59 ⫾ 0.03%) and increased
(1.43 ⫾ 0.11%) respectively, after LGF treatment with respect to the corresponding CSE group. However, there were not significant differences in the percentage of neutrophils in the blood samples of mice treated with LGF after establishing emphysema. Leukocyte population was identified by flow cytometry analysis based on their characteristic properties shown in the FSC and SSC. Representative gating was set for CD3⫹ on T-lymphocytes, Ly6B.2hi on neutrophils, and F4/80⫹ on monocytes from BAL fluid and peripheral blood of mice (Figs. 6B and Fig. 7B). LGF treatment reduces the serum TNF-␣ level after chronic CSE. Levels of the cytokine TNF-␣ were significantly greater in the peripheral blood of the CSE mice (362 ⫾ 25.93 pg/ml) than CTL animals (267 ⫾ 9.73 pg/ml). LGF treatment brought TNF-␣ blood levels closer to normal values (315 ⫾ 20.25 pg/ml) but not significantly (Fig. 8). DISCUSSION
In the present study, it is demonstrated for the first time the LGF therapeutic effect on chronic CSE in a AKR/J mouse model of COPD, improving indicators and parameters of systemic inflammation, physiology, and morphology of the lungs. In this regard, the main findings in this study are that mice treated with LGF after chronic CSE (6 mo) exhibit the following modifications compared with control animals: 1) improved lung function impairment, 2) reduced alveolar airspace enlargement, 3) decreased chronic CSEmediated lung tissue remodeling enzyme activity, 4) reduced inflammatory cell infiltrate in BAL fluid, and 5) changed circulating leukocyte profile in peripheral blood, with decreasing T-lymphocyte population, tending to reduce neutrophil infiltration, and increasing monocyte population.
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00293.2013 • www.ajplung.org
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Fig. 3. Effect of LGF treatment on chronic CSEmediated matrix metalloproteinase (MMP) activity. A: LGF treatment decreases the chronic CSE-mediated lung tissue remodeling enzyme activity (arbitrary units from the control group, shown as per unit) in the lungs of the CSE group (solid bar) and CSE ⫹ LGF group (shaded bar) in AKR/J mice compared with those in CTL group (open bar). Data are expressed as means ⫾ SE (n ⫽ 4/group, *P ⬍ 0.05). B: lung imaging in vivo of 1 representative mouse of each group. Male mice were anesthetized and imaged using an IVIS Imaging System after 6 mo of CSE. The entire efficiency signal from the thorax was measured with Living Image Software by using spectral unmixing technique.
Considering these data, we suggest that LGF could be a relevant anti-inflammatory and lung structure and function regenerative molecule, particularly in situations of lung injury induced by CSE. COPD is associated with systemic effects, including reduced body weight gain and altered circulating leukocytes and plasma TNF-␣ levels. CSE mice gained body weight, although to a lesser extent compared with CTL animals, up to the third month of exposure. Although not specifically quantified, food intake between CSE and CTL rodents was similar, even after 3 mo of study (animals in both groups were always fed ad libitum, receiving an identical amount of food/day per animal in the cages). The present findings are in line with previous
investigations, where animals chronically exposed to cigarette smoke also underwent a significant reduction in body weight gain (2, 20). The long-term intraperitoneal administration of LGF doses (1.7 g/animal) in AKR/J mice was well tolerated, with no toxicity in terms of body weight gain. In summary, AKR/J mice chronic exposure to cigarette smoke induces lung emphysema concomitantly with greater modifications on body weight gain, total cell count in BAL fluid, MMP activity in living mice, physiological and morphological lung parameters, serum TNF-␣ levels, and change in the profile of circulating leukocytes. Treatment with LGF seems to reverse all these parameters studied, except the loss of body weight and TNF-␣ level, although the latter if it tends to decrease.
Fig. 4. Analysis of MMP gene expression by real-time PCR. The MMP-2 and MMP-9 mRNA levels relative to 18S rRNA are shown. MMP-2 increases in CSE group (solid bar), and LGF treatment decreases the MMP-9 expression in the CSE ⫹ LGF group (shaded bar) compared with CTL group (open bar). Results are expressed as means ⫾ SE (n ⫽ 6/group, *P ⬍ 0.05) in % relative to nonexposed and nontreated mice that were arbitrarily assigned a value of 1%.
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00293.2013 • www.ajplung.org
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Fig. 5. Effect of LGF treatment on inflammatory cell infiltrate in the bronchoalveolar lavage (BAL) fluid of emphysematous mice. LGF treatment (shaded bar) decreases the inflammatory cell infiltrate in the BAL fluid after chronic CSE (solid bar) compared with CTL group (open bar). The data represent means ⫾ SE of the total cell number (⫻103/ml) in the BAL fluid of different groups (n ⫽ 8/group, *P ⬍ 0.05).
In the current investigation, AKR/J mice exposed to cigarette smoke for 6 mo exhibited a significant increase in the CL (28%) and in the LM, alveolar airspace enlargement (12%), suggesting the development of emphysema in these animals compared with nonexposed control animals. These findings are in agreement with other previous studies (20). Both of these
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parameters were reduced with the LGF treatment by 50% and 11%, respectively. The presence of inflammation after chronic CSE in AKR/J mice was confirmed by increased inflammatory cell infiltrate in BAL fluid and by increased lung tissue remodeling enzyme activity measured in living mice. These findings are in agreement with other previous studies (47), but the novelty is the technique used to measure lung protease activity in vivo. Enzyme-based activatable probes for fluorescence-mediated imaging has proven valuable for the detection of tumor-associated lysosomal protease activity in an animal model (5, 45), and we have recently demonstrated that MMP-activatable probe can be used to detect MMP activity in the lung tissue after acute CSE (33). With the help of an activatable sensor that reports activity, we have demonstrated in the present study that MMP activity, visualized by fluorescence molecular imaging in the lung of living mice, is significantly decreased in LGF-treated mice after smoking-induced emphysema. The data presented herein serve to further evince molecular imaging as a technology highly appropriate for chronic lung inflammatory research. LGF treatment appears to reverse gene expression of MMP-9 in the lung tissue but not MMP-2. MMP-9 is produced mainly by inflammatory cells and could be rather linked to inflammatory process-induced tissue remodeling (10, 19, 40). However, MMP-2 is synthesized by structural cells and is more associated with the impaired tissue remodeling, leading to pathological collagen deposition and interstitial fibrosis. We consider that decreasing MMP-9 participates in early changes, as a consequence of the anti-inflammatory effect of LGF and MMP2 could possibly decrease later, while the process continues. We notice that the reversibility changes were studied only 15 days after LGF administration.
Fig. 6. Effect of LGF treatment on the leukocyte population profile in BAL fluid of CSE mice. A: LGF treatment changes the leukocyte profile in BAL fluid after chronic CSE. Shown are the percentages of T-lymphocytes, neutrophils, and alveolar macrophages in the BAL fluid of the CSE group (solid bar) and CSE ⫹ LGF group (shaded bar) compared with those in CTL group (open bar). Data are expressed as means ⫾ SE (percentage of cells, per unit from the control group); *P ⬍ 0.05; n ⫽ 6/group. B: representative gating was set for CD3⫹ on T-lymphocytes (red), Ly6B.2hi on neutrophils (yellow), and F4/80⫹ on alveolar macrophages (blue) from BAL fluid of mice. FSC, forward scatter plot; SSC, side scatter plot. AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00293.2013 • www.ajplung.org
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Fig. 7. Effect of LGF treatment on the circulating leukocyte population profile in peripheral blood of CSE mice. A: LGF treatment changes the leukocyte profile in peripheral blood after chronic CSE. Shown are the percentages of T-lymphocytes, neutrophils, and monocytes in the peripheral blood samples of the CSE group (solid bar) and CSE ⫹ LGF group (shaded bar) compared with those in CTL group (open bar). Data are expressed as means ⫾ SE (percentage of cells, per unit from the control group); *P ⬍ 0.05; n ⫽ 8/group. B: representative gating was set for CD3⫹ on T-lymphocytes (red), Ly6B.2hi on neutrophils (yellow), and F4/80⫹ on monocytes (blue) from peripheral blood of mice.
Systemic inflammation in COPD is also characterized by cytokines systemically implicated in inflammatory cell recruitment and airway remodeling. Our data suggest that animals with chronic airflow limitation had significantly raised levels of various systemic inflammatory markers like circulating leukocytes (specifically T-cells and neutrophils) and serum TNF-␣ level, indicating that persistent systemic inflammation is present in AKR/J. LGF treatment decreases T-lymphocyte population in peripheral blood, what seems to be in agreement with a recently published article that shows that T-cell depletion protects against alveolar destruction attributable to chronic CSE in mice (37). It is possible in some cases that the inflammatory process may “spill” over into the systemic circulation, promoting a generalized inflammatory reaction. Our findings are consistent with systemic inflammation present in some patients with COPD (1) associated with an increased influx of T-cells into the airway (6, 28, 31, 37). This finding may explain, at least in part, the high prevalence of systemic complications, such as cachexia, osteoporosis, and cardiovascular diseases among patients with COPD. Future studies are needed to determine whether attenuation of the systemic inflammatory process can modify the risk of these complications in COPD. In view of the results obtained in this study, what seems clear is that LGF treatment promotes tissue regeneration, probably by promoting cell proliferation (7, 18, 39, 42) of the lung parenchyma. Maybe the LGF stimulates cellular antioxidant response (8, 9) or decreases cellular apoptotic processes (13). However, further studies are necessary to get to know the mechanism by which it acts. In summary, there is now a large
body of evidence to indicate that LGF treatment reverses emphysema previously established in a chronic CSE mouse model, normalizing the physiological and morphological data and levels of various systemic inflammatory markers, which may have important pathophysiological and therapeutic implications for subjects with stable COPD.
Fig. 8. Effect of LGF treatment on serum TNF-␣ level after chronic CSE. LGF treatment decreases the serum TNF-␣ level after chronic CSE. Mean values of the TNF-␣ expressed as pg/ml in the blood samples of the CSE group (solid bar) and CSE ⫹ LGF group (shaded bar) in AKR/J mice compared with those in CTL group (open bar). Levels of the cytokine TNF-␣ were significantly greater in the blood of CSE mice compared with CTL animals. Data are expressed as means ⫾ SE (n ⫽ 8/group, **P ⬍ 0.05).
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00293.2013 • www.ajplung.org
LGF REVERSES EMPHYSEMA MOUSE MODEL ACKNOWLEDGMENTS We thank D. Angelos Kyriazis, Institute of Biofunctional Studies, Complutense University of Madrid - Spain, for his help with the P-V curve acquisition with the ventilator. GRANTS This study was supported by the Spanish Society of Pulmonology and Thoracic Surgery (SEPAR-139). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: S.P.-R. and G.P.-B. conception and design of research; S.P.-R., L.d.P.-N., Á.G.-M., and R.T.-E. analyzed data; S.P.-R., L.d.P.N., Á.G.-M., R.T.-E., and J.D.-G. interpreted results of experiments; S.P.-R., L.d.P.-N., and G.P.-B. drafted manuscript; S.P.-R., J.D.-G., and N.G.-M. edited and revised manuscript; S.P.-R., N.G.-M., and G.P.-B. approved final version of manuscript; L.d.P.-N., Á.G.-M., and R.T.-E. performed experiments; L.d.P.-N., Á.G.-M., and R.T.-E. prepared figures. REFERENCES 1. Agusti A, Edwards LD, Rennard SI, MacNee W, Tal-Singer R, Miller BE, Vestbo J, Lomas DA, Calverley PM, Wouters E, Crim C, Yates JC, Silverman EK, Coxson HO, Bakke P, Mayer RJ, Celli B. Persistent systemic inflammation is associated with poor clinical outcomes in COPD: a novel phenotype. PLoS One 7: e37483, 2012. 2. Ardite E, Peinado VI, Rabinovich RA, Fernandez-Checa JC, Roca J, Barbera JA. Systemic effects of cigarette smoke exposure in the guinea pig. Respir Med 100: 1186 –1194, 2006. 3. Barnes PJ, Hansel TT. Prospects for new drugs for chronic obstructive pulmonary disease. Lancet 364: 985–996, 2004. 4. Barreiro E, del Puerto-Nevado L, Puig-Vilanova E, Perez-Rial S, Sanchez F, Martinez-Galan L, Rivera S, Gea J, Gonzalez-Mangado N, Peces-Barba G. Cigarette smoke-induced oxidative stress in skeletal muscles of mice. Respir Physiol Neurobiol 182: 9 –17, 2012. 5. Bremer C, Tung CH, Weissleder R. In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat Med 7: 743–748, 2001. 6. Brozyna S, Ahern J, Hodge G, Nairn J, Holmes M, Reynolds PN, Hodge S. Chemotactic mediators of Th1 T-cell trafficking in smokers and COPD patients. COPD 6: 4 –16, 2009. 7. Conde MV, Gonzalez MC, Quintana-Villamandos B, Abderrahim F, Briones AM, Condezo-Hoyos L, Regadera J, Susin C, Gomez de Diego JJ, Delgado-Baeza E, Diaz-Gil JJ, Arribas SM. Liver growth factor treatment restores cell-extracellular matrix balance in resistance arteries and improves left ventricular hypertrophy in SHR. Am J Physiol Heart Circ Physiol 301: H1153–H1165, 2011. 8. Condezo-Hoyos L, Abderrahim F, Conde MV, Susin C, Diaz-Gil JJ, Gonzalez MC, Arribas SM. Antioxidant activity of liver growth factor, a bilirubin covalently bound to albumin. Free Radic Biol Med 46: 656 –662, 2009. 9. Condezo-Hoyos L, Arribas SM, Abderrahim F, Somoza B, Gil-Ortega M, Diaz-Gil JJ, Conde MV, Susin C, Gonzalez MC. Liver growth factor treatment reverses vascular and plasmatic oxidative stress in spontaneously hypertensive rats. J Hypertens 30: 1185–1194, 2012. 10. Chakrabarti S, Patel KD. Matrix metalloproteinase-2 (MMP-2) and MMP-9 in pulmonary pathology. Exp Lung Res 31: 599 –621, 2005. 11. Churg A, Wang RD, Tai H, Wang X, Xie C, Dai J, Shapiro SD, Wright JL. Macrophage metalloelastase mediates acute cigarette smokeinduced inflammation via tumor necrosis factor-alpha release. Am J Respir Crit Care Med 167: 1083–1089, 2003. 12. Diaz-Gil JJ, Escartin P, Garcia-Canero R, Trilla C, Veloso JJ, Sanchez G, Moreno-Caparros A, Enrique de Salamanca C, Lozano R, Gavilanes JG, García-Segura JM. Purification of a liver DNA-synthesis promoter from plasma of partially hepatectomized rats. Biochem J 235: 49 –55, 1986. 13. Diaz-Gil JJ, Garcia-Monzon C, Rua C, Martin-Sanz P, Cereceda RM, Miquilena-Colina ME, Machin C, Fernandez-Martinez A, GarciaCanero R. Liver growth factor antifibrotic activity in vivo is associated with a decrease in activation of hepatic stellate cells. Histol Histopathol 24: 473–479, 2009.
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AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00293.2013 • www.ajplung.org
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