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
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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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.
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
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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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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LGF REVERSES EMPHYSEMA MOUSE MODEL
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).
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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.
LGF REVERSES EMPHYSEMA MOUSE MODEL
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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.
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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.
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LGF REVERSES EMPHYSEMA MOUSE MODEL
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%.
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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.
LGF REVERSES EMPHYSEMA MOUSE MODEL
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
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.
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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).
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LGF REVERSES EMPHYSEMA MOUSE MODEL
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).
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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.
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
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.
14. Diaz-Gil JJ, Munoz J, Albillos A, Rua C, Machin C, Garcia-Canero R,
Cereceda RM, Guijarro MC, Trilla C, Escartin P. Improvement in liver
fibrosis, functionality and hemodynamics in CCI4-cirrhotic rats after
injection of the Liver Growth Factor. J Hepatol 30: 1065–1072, 1999.
15. Diaz Gil JJ, Rua C, Machin C, Cereceda RM, Garcia-Canero R, de
Foronda M, Perez de Diego J, Trilla C, Escartin P. Hepatic growth
induced by injection of the liver growth factor into normal rats. Growth
Regul 4: 113–122, 1994.
16. Gan WQ, Man SF, Senthilselvan A, Sin DD. Association between
chronic obstructive pulmonary disease and systemic inflammation: a
systematic review and a meta-analysis. Thorax 59: 574 –580, 2004.
17. Gonzalo-Gobernado R, Calatrava-Ferreras L, Reimers D, Herranz
AS, Rodriguez-Serrano M, Miranda C, Jimenez-Escrig A, Diaz-Gil
JJ, Bazan E. Neuroprotective activity of peripherally administered liver
growth factor in a rat model of Parkinson’s disease. PLoS One 8: e67771,
2013.
18. Gonzalo-Gobernado R, Reimers D, Herranz AS, Diaz-Gil JJ, Osuna
C, Asensio MJ, Baena S, Rodriguez-Serrano M, Bazan E. Mobilization
of neural stem cells and generation of new neurons in 6-OHDA-lesioned
rats by intracerebroventricular infusion of liver growth factor. J Histochem
Cytochem 57: 491–502, 2009.
19. Gueders MM, Foidart JM, Noel A, Cataldo DD. Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs in the respiratory tract:
potential implications in asthma and other lung diseases. Eur J Pharmacol
533: 133–144, 2006.
20. Guerassimov A, Hoshino Y, Takubo Y, Turcotte A, Yamamoto M,
Ghezzo H, Triantafillopoulos A, Whittaker K, Hoidal JR, Cosio MG.
The development of emphysema in cigarette smoke-exposed mice is strain
dependent. Am J Respir Crit Care Med 170: 974 –980, 2004.
21. Haller J, Hyde D, Deliolanis N, de Kleine R, Niedre M, Ntziachristos
V. Visualization of pulmonary inflammation using noninvasive fluorescence molecular imaging. J Appl Physiol 104: 795–802, 2008.
22. Harris RS, Hess DR, Venegas JG. An objective analysis of the pressurevolume curve in the acute respiratory distress syndrome. Am J Respir Crit
Care Med 161: 432–439, 2000.
23. Hauber HP, Karp D, Goldmann T, Vollmer E, Zabel P. Comparison of
the effect of LPS and PAM3 on ventilated lungs. BMC Pulm Med 10: 20,
2010.
24. Hautamaki RD, Kobayashi DK, Senior RM, Shapiro SD. Requirement
for macrophage elastase for cigarette smoke-induced emphysema in mice.
Science 277: 2002–2004, 1997.
25. Irvin CG, Bates JH. Measuring the lung function in the mouse: the
challenge of size. Respir Res 4: 4, 2003.
26. Kyriazis A, Perez de Alejo R, Rodriguez I, Olsson LE, Ruiz-Cabello
JA. MRI and polarized gases compatible respirator and gas administrator
for the study of the small animal lung: volume measurement and control.
IEEE Trans Biomed Eng 57: 1745–1749, 2010.
27. Lagente V, Manoury B, Nenan S, Le Quement C, Martin-Chouly C,
Boichot E. Role of matrix metalloproteinases in the development of
airway inflammation and remodeling. Braz J Med Biol Res 38: 1521–1530,
2005.
28. Maeno T, Houghton AM, Quintero PA, Grumelli S, Owen CA,
Shapiro SD. CD8⫹ T Cells are required for inflammation and destruction
in cigarette smoke-induced emphysema in mice. J Immunol 178: 8090 –
8096, 2007.
29. Mahmood U, Tung CH, Bogdanov A, Jr., Weissleder R. Near-infrared
optical imaging of protease activity for tumor detection. Radiology 213:
866 –870, 1999.
30. Martinez-Galan L, del Puerto-Nevado L, Perez-Rial S, Diaz-Gil JJ,
Gonzalez-Mangado N, Peces-Barba G. [Liver growth factor improves
pulmonary fibrosis secondary to cadmium administration in rats]. Arch
Bronconeumol 46: 20 –26, 2010.
31. Motz GT, Eppert BL, Wesselkamper SC, Flury JL, Borchers MT.
Chronic cigarette smoke exposure generates pathogenic T cells capable of
driving COPD-like disease in Rag2-/- mice. Am J Respir Crit Care Med
181: 1223–1233, 2010.
32. Perez-Crespo M, Pericuesta E, Perez-Cerezales S, Arenas MI, Lobo
MV, Diaz-Gil JJ, Gutierrez-Adan A. Effect of liver growth factor on
both testicular regeneration and recovery of spermatogenesis in busulfantreated mice. Reprod Biol Endocrinol 9: 21, 2011.
33. Perez-Rial S, Del Puerto-Nevado L, Gonzalez-Mangado N, PecesBarba G. Early detection of susceptibility to acute lung inflammation by
molecular imaging in mice exposed to cigarette smoke. Mol Imaging 10:
398 –405, 2011.
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00293.2013 • www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 18, 2017
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.
L725
L726
LGF REVERSES EMPHYSEMA MOUSE MODEL
41. Shapiro SD, Senior RM. Matrix metalloproteinases, matrix degradation
and more. Am J Respir Cell Mol Biol 20: 1100 –1102, 1999.
42. Somoza B, Abderrahim F, Gonzalez JM, Conde MV, Arribas SM,
Starcher B, Regadera J, Fernandez-Alfonso MS, Diaz-Gil JJ, Gonzalez MC. Short-term treatment of spontaneously hypertensive rats
with liver growth factor reduces carotid artery fibrosis, improves
vascular function, and lowers blood pressure. Cardiovasc Res 69:
764 –771, 2006.
43. Stewart RD, Baretta ED, Platte LR, Stewart EB, Kalbfleisch JH, Van
Yserloo B, Rimm AA. Carboxyhemoglobin levels in American blood
donors. JAMA 229: 1187–1195, 1974.
44. Venegas JG, Harris RS, Simon BA. A comprehensive equation for
the pulmonary pressure-volume curve. J Appl Physiol 84: 389 –395,
1998.
45. Weissleder R, Tung CH, Mahmood U, Bogdanov A, Jr. In vivo imaging
of tumors with protease-activated near-infrared fluorescent probes. Nat Biotechnol 17: 375–378, 1999.
46. Xu H, Rice BW. In-vivo fluorescence imaging with a multivariate
curve resolution spectral unmixing technique. J Biomed Opt 14:
064011, 2009.
47. Yao H, Edirisinghe I, Rajendrasozhan S, Yang SR, Caito S, Adenuga
D, Rahman I. Cigarette smoke-mediated inflammatory and oxidative
responses are strain-dependent in mice. Am J Physiol Lung Cell Mol
Physiol 294: L1174 –L1186, 2008.
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00293.2013 • www.ajplung.org
Downloaded from http://ajplung.physiology.org/ by 10.220.33.6 on June 18, 2017
35. Perez-Rial S, Del Puerto-Nevado L, Terron-Exposito R, Giron-Martinez A, Gonzalez-Mangado N, Peces-Barba G. Role of recently migrated monocytes in cigarette smoke-induced lung inflammation in different strain of mice. PLoS One 8: e72975, 2013.
36. Perez de Alejo R, Ruiz-Cabello J, Villa P, Rodríguez I, Perez-Sanchez J,
Peces-Barba Romero G, Cortijo M. A fully MRI-compatible animal ventilator for special-gas mixing applications. Magn Res 26B: 93–103, 2005.
37. Podolin PL, Foley JP, Carpenter DC, Bolognese BJ, Logan GA, Long
E 3rd, Harrison OJ, Walsh PT. T cell depletion protects against alveolar
destruction due to chronic cigarette smoke exposure in mice. Am J Physiol
Lung Cell Mol Physiol 304: L312–L323, 2013.
38. Reimers D, Herranz AS, Diaz-Gil JJ, Lobo MV, Paino CL, Alonso R,
Asensio MJ, Gonzalo-Gobernado R, Bazan E. Intrastriatal infusion of
liver growth factor stimulates dopamine terminal sprouting and partially
restores motor function in 6-hydroxydopamine-lesioned rats. J Histochem
Cytochem 54: 457–465, 2006.
39. Reimers D, Osuna C, Gonzalo-Gobernado R, Herranz AS, Diaz-Gil JJ,
Jimenez-Escrig A, Asensio MJ, Miranda C, Rodriguez-Serrano M, Bazan E.
Liver growth factor promotes the survival of grafted neural stem cells in a rat
model of Parkinson’s disease. Curr Stem Cell Res Ther 7: 15–25, 2012.
40. Segura-Valdez L, Pardo A, Gaxiola M, Uhal BD, Becerril C, Selman
M. Upregulation of gelatinases A and B, collagenases 1 and 2, and
increased parenchymal cell death in COPD. Chest 117: 684 –694, 2000.