Efectos terapéuticos del factor de crecimiento de hígado (LGF) en un modelo de enfisema experimental inducido por la exposición crónica al humo del tabaco

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 26/06/2014El desarrollo de enfisema es un aspecto característico de la enfermedad pulmonar obstructiva crónica (EPOC), siendo el humo de tabaco el principal factor de riesgo. Actualmente, el tratamiento de la EPOC consiste en la administración de broncodilatadores y corticoesteroides para controlar la función pulmonar, los síntomas y las exacerbaciones, pero no existen terapias efectivas dirigidas a revertir la progresión de la enfermedad. El factor de crecimiento de hígado (LGF) es un complejo albúmina‐bilirrubina con propiedades mitogénicas, cuyos efectos terapéuticos han sido previamente descritos en otros modelos animales de enfermedades como Parkinson, daño hepático, degeneración testicular, ateroesclerosis, enfermedades cardiovasculares o hipertensión. Para evaluar los efectos del LGF en un modelo de enfisema, analizamos parámetros morfométricos y funcionales a nivel pulmonar, así como la actividad metaloproteásica y diversos marcadores de daño y de reparación tisular como VEGF, PCNA, 3‐NT y Nrf2 en ratones C57BL6/j expuestos de forma crónica al humo de tabaco y tratados posteriormente con una dosis adecuada de LGF por vía intraperitoneal. Los ratones expuestos al humo de tabaco presentaron un agrandamiento significativo de los espacios alveolares (Lm), mayor área interna alveolar (AIA) y pérdida de función pulmonar que correlacionó con una mayor actividad metaloproteásica, un incremento de la expresión de 3‐NT y un descenso de la expresión de VEGF. Por el contrario, aquellos ratones expuestos al humo de tabaco y tratados posteriormente con LGF mostraron una mejoría del daño pulmonar así como síntomas de recuperación de función pulmonar que correlacionó con una menor actividad metaloproteásica, un descenso en la expresión de 3‐NT y un incremento en la expresión de VEGF, PCNA y Nrf2. Por lo tanto, este estudio aporta evidencias de que el LGF presenta efectos terapéuticos en un modelo de enfisema previamente establecido, postulándose como una estrategia prometedora para revertir la progresión de la EPOC en un futuro.Emphysema is a major component of Chronic Obstructive Pulmonary Disease (COPD), considering cigarette smoke (CS) as the main risk factor. Currently, treatment of COPD is based on the administration of bronchodilators and corticosteroids to control the lung function, the symptoms and exacerbations, but there are no effective therapies directed to reverse the progression of the disease. Liver growth factor (LGF) is an albumin‐bilirubin complex with mitogenic properties, whose therapeutic effects has been previously reported in several rodent models such as injured liver, Parkinson`s disease, testis degeneration, atherosclerosis, heart or hypertension. To address the therapeutic effect of LGF, morphometric and lung function parameters, matrix metalloproteinase (MMP) activity and the expression of several markers such as VEGF, PCNA, 3‐NT and Nrf2 were assessed in air‐exposed and CS‐exposed C57BL/6j male mice with and without intraperitoneal (i.p.) injection of LGF administered after longterm cigarette smoke exposure (CSE). CS‐exposed mice presented a significant enlargement of alveolar spaces (Lm), higher alveolar internal area (AIA) and loss of lung function that correlated with higher MMP activity, higher expression of 3‐NT and lower expression of VEGF. CS‐exposed mice and then i.p. injected with LGF, showed an amelioration of emphysema and improved lung function that correlated with lower MMP activity and 3‐NT expression, and higher levels of VEGF, PCNA and Nrf2. Thus, this study provides evidence that LGF administration produced therapeutic effects once the lung damage was established, postulated as a promising strategy to revert the progression of COPD in the future

Seeding and Growth of β-Amyloid Aggregates upon Interaction with Neuronal Cell Membranes

In recent years, the prevalence of amyloid neurodegenerative diseases such as Alzheimer’s disease (AD) has significantly increased in developed countries due to increased life expectancy. This amyloid disease is characterized by the presence of accumulations and deposits of β-amyloid peptide (Aβ) in neuronal tissue, leading to the formation of oligomers, fibers, and plaques. First, oligomeric intermediates that arise during the aggregation process are currently thought to be primarily responsible for cytotoxicity in cells. This work aims to provide further insights into the mechanisms of cytotoxicity by studying the interaction of Aβ aggregates with Neuro-2a (N2a) neuronal cells and the effects caused by this interaction. For this purpose, we have exploited the advantages of advanced, multidimensional fluorescence microscopy techniques to determine whether different types of Aβ are involved in higher rates of cellular toxicity, and we measured the cellular stress caused by such aggregates by using a fluorogenic intracellular biothiol sensor. Stress provoked by the peptide is evident by N2a cells generating high levels of biothiols as a defense mechanism. In our study, we demonstrate that Aβ aggregates act as seeds for aggregate growth upon interacting with the cellular membrane, which results in cell permeability and damage and induces lysis. In parallel, these damaged cells undergo a significant increase in intracellular biothiol levels.Spanish Ministerio de Ciencia, Innovacion y Universidades CTQ2014-56370-R CTQ2017-86568-R CTQ2017-86125-PSpanish Agencia Estatal de InvestigacionEuropean Union (EU

Biografía y Contribución Científica de Simón de Rojas Clemente

Peer Reviewe

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

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

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