Proliferative activity of liver growth factor is associated with an improvement of cigarette smoke-induced emphysema in mice.

Cigarette smoke (CS)-induced emphysema is a major component of chronic obstructive pulmonary disease (COPD). COPD treatment is based on the administration of bronchodilators and corticosteroids to control symptoms and exacerbations, however, to date, there are no effective therapies to reverse disease progression. Liver growth factor (LGF) is an albumin-bilirubin complex with mitogenic properties, whose therapeutic effects have previously been reported in a model of emphysema and several rodent models of human disease. To approach the therapeutic effect of LGF in a model of previously established emphysema, morphometric and lung function parameters, matrix metalloproteinase (MMP) activity and the expression of several markers, such as VEGF, PCNA, 3NT and Nrf2, were assessed in air-exposed and CS-exposed C57BL/6J male mice with and without intraperitoneal (i.p.) injection of LGF. CS-exposed mice presented a significant enlargement of alveolar spaces, higher alveolar internal area and loss of lung function that correlated with higher MMP activity, higher expression of 3NT and lower expression of VEGF. CS-exposed mice injected with LGF, showed an amelioration of emphysema and improved lung function, which correlated with lower MMP activity and 3NT expression and higher levels of VEGF, PCNA and Nrf2. Taken together, this study suggests that LGF administration ameliorates CS-induced emphysema, highlights the ability of LGF to promote alveolar cell proliferation and may be a promising strategy to revert COPD progression

Role of recently migrated monocytes in cigarette smoke-induced lung inflammation in different strain of mice.

This study investigates the role of proinflammatory monocytes recruited from blood circulation and recovered in bronchoalveolar lavage (BAL) fluid in mediating the lung damage in a model of acute cigarette smoke (CS)-induced lung inflammation in two strains of mice with different susceptibility to develop emphysema (susceptible -C57BL/6J and non susceptible -129S2/SvHsd). Exposure to whole-body CS for 3 consecutive research cigarettes in one single day induced acute inflammation in the lung of mice. Analysis of BAL fluid showed more influx of recently migrated monocytes at 72 h after CS-exposition in susceptible compared to non susceptible mice. It correlated with an increase in MMP-12 and TNF-α protein levels in the lung tissue, and with an increment of NF-κB translocation to the nucleus measured by electrophoretic mobility shift assay in C57BL/6J mice. To determine the functional role of these proinflammatory monocytes in mediating CS-induced airway inflammation, alveolar macrophages and blood monocytes were transiently removed by pretreatment with intratracheal and intravenous liposome-encapsulated CL2MDP, given 2 and 4 days prior to CS exposure and their repopulation was studied. Monocytes/macrophages were maximally depleted 48 h after last liposome application and subsequently recently migrated monocytes reappeared in BAL fluid of susceptible mice at 72 h after CS exposure. Recently migrated monocytes influx to the lung correlated with an increase in the MMP-12 protein level in the lung tissue, indicating that the increase in proinflammatory monocytes is associated with a major tissue damaging. Therefore our data confirm that the recruitment of proinflammatory recently migrated monocytes from the blood are responsible for the increase in MMP-12 and has an important role in the pathogenesis of lung disease induced by acute lung inflammation. These results could contribute to understanding the different susceptibility to CS of these strains of mice

Effect of CS exposure on the absolute number of cells in BAL fluid.

<p>Increased total number of cells after CS-exposure in BAL fluid of susceptible mouse strain was found. Total cells in BAL fluid from smoke-exposed group (▪) at 24, 48 and 72 h after CS exposure in C57BL/6J (susceptible) and 129S2/SvHsd (non susceptible) mice compared to those in the air exposed group (□) and expressed the mean ± SEM; *p<0.05; n = 8/group.</p

Time-course leukocytes population profile in BAL fluid after CS exposure.

<p>(<b>A</b>) Percentage of neutrophils, recently migrated monocytes and recently differentiated alveolar macrophages in BAL fluid from smoke-exposed group (▪) at 24, 48 and 72 h after CS exposure in C57BL/6J susceptible and 129S2/SvHsd non susceptible mice compared to those in the air exposed group (□) and expressed the mean ± SEM; *p<0.05; n = 8/group. (<b>B</b>) Leukocytes population was identified by flow cytometry analysis based on their characteristic properties shown in the forward scatter (FSC) and sideward scatter (SSC). Representative gating was set for Ly6B.2^hi on neutrophils (yellow), Ly6B.2⁺ on recently migrated monocytes (pink) and F4/80^hi on recently differentiated alveolar macrophages (blue) from BAL fluid of each strain of mice.</p

Leukocytes repopulation profile in BAL fluid induced by CS exposure after transient monocyte/macrophage.

<p>Effect of CS exposure on cell repopulation after CL₂MDP treatment was studied by flow cytometry analysis in BAL fluid from susceptible mice. Percentage of neutrophils, recently migrated monocytes and recently differentiated alveolar macrophages in BAL fluid from smoke-exposed group (▪) at 72 h after CS exposure and pretreatment with CL₂MDP in C57BL/6J-susceptible and 129S2/SvHsd-non susceptible mice compared with their respective CL₂MDP -treated air exposed group () and expressed the mean ± SEM; *p<0.05, **p<0.01; n = 8/group.</p

LGF ameliorates oxidative stress.

<p>Bars represent (<b>A</b>) 3NT and (<b>B</b>) Nrf2 levels estimated by western blot. Data were normalized with tubulin. * <i>P</i><0.05 <i>vs</i>. air-exposed mice; ¥ <i>P</i><0.05 <i>vs</i>. CS-exposed mice (n = 5 per group). Data are presented as mean ± SEM.</p

Alveolar space enlargement and lung function.

<p>(<b>A</b>) Histological sections from the lungs stained with H&E (n = 5 per group). Scale bars = 50 µm. Mean chord length (L_m) (<b>B</b>) and the mean of the alveolar internal area (<b>C</b>) of alveoli in the lungs of air-exposed mice untreated (C) and treated with LGF (C+LGF) and CS-exposed mice untreated (CSE) and treated with LGF (CSE+LGF). (<b>D</b>) Distribution analysis of random intercepts obtained from measurements on randomly sampled images on linear scale (n = 5 per group). (<b>E</b>) V_max was used to evaluate lung function of air-exposed mice (C), CS-exposed mice (CSE) and CS-exposed mice post-treated with LGF (CSE+LGF) (n = 5 per group). * <i>P</i><0.05 and ** <i>P</i><0.01 <i>vs.</i> air-exposed mice; ¥ <i>P</i><0.05 and ¥¥ <i>P</i><0.01 <i>vs.</i> CS-exposed mice. Data are presented as mean ± SEM.</p

Tissue levels of VEGF and PCNA.

<p>VEGF levels were estimated by (<b>A</b>) western blot and (<b>B</b>) ELISA. (<b>C</b>) PCNA levels estimated by western blot. Data were normalized with tubulin. (<b>D</b>) Representative images of PCNA immunohistochemistry staining in the nucleus is shown for CS-exposed mice (CSE) and CS-exposed and LGF-treated mice (CSE+LGF). Scale bars = 50 µm. LGF promoted an increase of PCNA+ cells as showed in the bar graph. ** <i>P</i><0.01 <i>vs.</i> air-exposed mice; ¥ <i>P</i><0.05 <i>vs.</i> CS-exposed mice (n = 5 per group). Data are presented as mean ± SEM.</p

MMP activity.

<p>(<b>A</b>) In vivo MMP activity by FMI. Images were captured using the IVIS Imaging System. The entire efficiency signal from the thorax was measured with Living Image software using a spectral unmixing technique. One representative mouse of each group is shown. (<b>B</b>) Bars represent MMP activity in the lungs from air-exposed group (C), CS-exposed group (CSE) and LGF-treated CS-exposed group (CSE+LGF). The units used for the analysis of images were the ratio between emission and excitation light (referred to as efficiency). (<b>C</b>) Representative gelatin zymography indicating the activities of MMP-9 (<b>D</b>) and MMP-2 (<b>E</b>) in normal lungs and in lungs tissues from mice expose to CS and then non-treated or treated with LGF. Administration of LGF attenuated MMP-9 activity in CS-exposed mice. However, the MMP-2 activity was not attenuated by LGF administration. The expression of MMP-9 (<b>F</b>), MMP-2 (<b>G</b>), TIMP-1 (<b>H</b>) and TIMP-2 (<b>I</b>) mRNA relative to 18S rRNA were analyzed by real-time PCR. Results were expressed in % relative to non-exposed and non-treated mice that were arbitrarily assigned a value of 100%. * <i>P</i><0.05 and ** <i>P</i><0.01 <i>vs.</i> air-exposed mice; ¥ <i>P</i><0.05 and ¥¥ <i>P</i><0.01 vs. CS-exposed mice (n = 5 per group). Data are presented as mean ± SEM.</p

Confirmation that macrophage depletion in BAL fluid induced by CL₂MDP is effective.

<p>Effect on macrophage depletion after CL₂MDP treatment was studied by flow cytometry analysis in BAL fluid from C57BL/6J-susceptible and 129S2/SvHsd-resistant non-smoke-exposed mice (control group). Percentage of recently migrated monocytes and recently differentiated alveolar macrophages in BAL fluid from CL₂MDP-treated control group () at 48 h after treatment compared to those in the PBS-treated control group (□) and expressed the mean ± SEM; *p<0.05, **p<0.01; n = 7–8/group.</p