Gisele A. Padilha

Bosutinib Therapy Ameliorates Lung Inflammation and Fibrosis in Experimental Silicosis

Silicosis is an occupational lung disease for which no effective therapy exists. We hypothesized that bosutinib, a tyrosine kinase inhibitor, might ameliorate inflammatory responses, attenuate pulmonary fibrosis, and thus improve lung function in experimental silicosis. For this purpose, we investigated the potential efficacy of bosutinib in the treatment of experimental silicosis induced in C57BL/6 mice by intratracheal administration of silica particles. After 15 days, once disease was established, animals were randomly assigned to receive DMSO or bosutinib (1 mg/kg/dose in 0.1 mL 1% DMSO) by oral gavage, twice daily for 14 days. On day 30, lung mechanics and morphometry, total and differential cell count in alveolar septa and granuloma, levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-4, transforming growth factor (TGF)-β, and vascular endothelial growth factor in lung homogenate, M1 and M2 macrophages, total leukocytes, and T cells in BALF, lymph nodes, and thymus, and collagen fiber content in alveolar septa and granuloma were analyzed. In a separate in vitro experiment, RAW264.7 macrophages were exposed to silica particles in the presence or absence of bosutinib. After 24 h, gene expressions of arginase-1, IL-10, IL-12, inducible nitric oxide synthase (iNOS), metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1, and caspase-3 were evaluated. In vivo, in silicotic animals, bosutinib, compared to DMSO, decreased: (1) fraction area of collapsed alveoli, (2) size and number of granulomas, and mononuclear cell granuloma infiltration; (3) IL-1β, TNF-α, IFN-γ, and TGF-β levels in lung homogenates, (4) collagen fiber content in lung parenchyma, and (5) viscoelastic pressure and static lung elastance. Bosutinib also reduced M1 cell counts while increasing M2 macrophage population in both lung parenchyma and granulomas. Total leukocyte, regulatory T, CD4+, and CD8+ cell counts in the lung-draining lymph nodes also decreased with bosutinib therapy without affecting thymus cellularity. In vitro, bosutinib led to a decrease in IL-12 and iNOS and increase in IL-10, arginase-1, MMP-9, and TIMP-1. In conclusion, in the current model of silicosis, bosutinib therapy yielded beneficial effects on lung inflammation and remodeling, therefore resulting in lung mechanics improvement. Bosutinib may hold promise for silicosis; however, further studies are required.

Therapeutic effects of LASSBio-596 in an elastase-induced mouse model of emphysema.

Emphysema is an intractable pulmonary disease characterized by an inflammatory process of the airways and lung parenchyma and ongoing remodeling process in an attempt to restore lung structure. There is no effective drug therapy that regenerates lung tissue or prevents the progression of emphysema; current treatment is aimed at symptomatic relief. We hypothesized that LASSBio-596, a molecule with potent anti-inflammatory and immunomodulatory effects, might reduce pulmonary inflammation and remodeling and thus improve lung function in experimental emphysema. Emphysema was induced in BALB/c mice by intratracheal administration of porcine pancreatic elastase (0.1 IU) once weekly during 4 weeks. A control group received saline using the same protocol. After the last instillation of saline or elastase, dimethyl sulfoxide, or LASSBio-596 were administered intraperitoneally, once daily for 8 days. After 24 h, in elastase-induced emphysema animals, LASSBio-596 yielded: (1) decreased mean linear intercept, hyperinflation and collagen fiber content, (2) increased elastic fiber content, (3) reduced number of M1 macrophages, (4) decreased tumor necrosis factor-α, interleukin-1β, interleukin-6, and transforming growth factor-β protein levels in lung tissue, and increased vascular endothelial growth factor. These changes resulted in increased static lung elastance. In conclusion, LASSBio-596 therapy reduced lung inflammation, airspace enlargement, and small airway wall remodeling, thus improving lung function, in this animal model of elastase-induced emphysema.

Characterization of a Mouse Model of Emphysema Induced by Multiple Instillations of Low-Dose Elastase

Many experimental models have been proposed to study the pathophysiological features of emphysema, as well as to search for new therapeutic approaches for acute or chronically injured lung parenchyma. We aimed to characterize an emphysema model induced by multiple instillations of elastase by tracking changes in inflammation, remodeling, and cardiac function after each instillation. Forty-eight C57BL/6 mice were randomly assigned across two groups. Emphysema (ELA) animals received 1, 2, 3, or 4 intratracheal instillations of pancreatic porcine elastase (PPE, 0.2 IU) with a 1-week interval between them. Controls (C) received saline following the same protocol. Before and after implementation of the protocol, animals underwent echocardiographic analysis. After the first instillation of PPE, the percentage of mononuclear cells in the lung parenchyma increased compared to C (p = 0.0001). The second instillation resulted in hyperinflated alveoli, increased mean linear intercept, and reduced elastic fiber content in lung parenchyma compared to C (p = 0.0197). Following the third instillation, neutrophils and collagen fiber content in alveolar septa and airways increased, whereas static lung elastance was reduced compared to C (p = 0.0094). After the fourth instillation, the percentage of M1 macrophages in lungs; levels of interleukin-1β (IL-1β), keratinocyte-derived chemokine, hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF); and collagen fiber content in the pulmonary vessel wall were increased compared to C (p = 0.0096). At this time point, pulmonary arterial hypertension was apparent, with increased diastolic right ventricular wall thickness. In conclusion, the initial phase of emphysema was characterized by lung inflammation with predominance of mononuclear cells, whereas at the late stage, impairment of pulmonary and cardiovascular functions was observed. This model enables analysis of therapies at different time points during controlled progression of emphysema. Accordingly, early interventions could focus on the inflammatory process, while late interventions should focus on restoring cardiorespiratory function.

Comparison between Variable and Conventional Volume-Controlled Ventilation on Cardiorespiratory Parameters in Experimental Emphysema.

Emphysema is characterized by loss of lung tissue elasticity and destruction of structures supporting alveoli and capillaries. The impact of mechanical ventilation strategies on ventilator-induced lung injury (VILI) in emphysema is poorly defined. New ventilator strategies should be developed to minimize VILI in emphysema. The present study was divided into two protocols: (1) characterization of an elastase-induced emphysema model in rats and identification of the time point of greatest cardiorespiratory impairment, defined as a high specific lung elastance associated with large right ventricular end-diastolic area; and (2) comparison between variable (VV) and conventional volume-controlled ventilation (VCV) on lung mechanics and morphometry, biological markers, and cardiac function at that time point. In the first protocol, Wistar rats (n = 62) received saline (SAL) or porcine pancreatic elastase (ELA) intratracheally once weekly for 4 weeks, respectively. Evaluations were performed 1, 3, 5, or 8 weeks after the last intratracheal instillation of saline or elastase. After identifying the time point of greatest cardiorespiratory impairment, an additional 32 Wistar rats were randomized into the SAL and ELA groups and then ventilated with VV or VCV (n = 8/group) [tidal volume (VT) = 6 mL/kg, positive end-expiratory pressure (PEEP) = 3 cmH2O, fraction of inspired oxygen (FiO2) = 0.4] for 2 h. VV was applied on a breath-to-breath basis as a sequence of randomly generated VT values (mean VT = 6 mL/kg), with a 30% coefficient of variation. Non-ventilated (NV) SAL and ELA animals were used for molecular biology analysis. The time point of greatest cardiorespiratory impairment, was observed 5 weeks after the last elastase instillation. At this time point, interleukin (IL)-6, cytokine-induced neutrophil chemoattractant (CINC)-1, amphiregulin, angiopoietin (Ang)-2, and vascular endothelial growth factor (VEGF) mRNA levels were higher in ELA compared to SAL. In ELA animals, VV reduced respiratory system elastance, alveolar collapse, and hyperinflation compared to VCV, without significant differences in gas exchange, but increased right ventricular diastolic area. Interleukin-6 mRNA expression was higher in VCV and VV than NV, while surfactant protein-D was increased in VV compared to NV. In conclusion, VV improved lung function and morphology and reduced VILI, but impaired right cardiac function in this model of elastase induced-emphysema.

Moderate Aerobic Training Improves Cardiorespiratory Parameters in Elastase-Induced Emphysema

Aim: We investigated the therapeutic effects of aerobic training on lung mechanics, inflammation, morphometry and biological markers associated with inflammation, and endothelial cell damage, as well as cardiac function in a model of elastase-induced emphysema. Methods: Eighty-four BALB/c mice were randomly allocated to receive saline (control, C) or 0.1 IU porcine pancreatic elastase (emphysema, ELA) intratracheally once weekly for 4 weeks. After the end of administration period, once cardiorespiratory impairment associated with emphysema was confirmed, each group was further randomized into sedentary (S) and trained (T) subgroups. Trained mice ran on a motorized treadmill, at moderate intensity, 30 min/day, 3 times/week for 4 weeks. Results: Four weeks after the first instillation, ELA animals, compared to C, showed: (1) reduced static lung elastance (Est,L) and levels of vascular endothelial growth factor (VEGF) in lung tissue, (2) increased elastic and collagen fiber content, dynamic elastance (E, in vitro), alveolar hyperinflation, and levels of interleukin-1β and tumor necrosis factor (TNF)-α, and (3) increased right ventricular diastolic area (RVA). Four weeks after aerobic training, ELA-T group, compared to ELA-S, was associated with reduced lung hyperinflation, elastic and collagen fiber content, TNF-α levels, and RVA, as well as increased Est,L, E, and levels of VEGF. Conclusion: Four weeks of regular and moderate intensity aerobic training modulated lung inflammation and remodeling, thus improving pulmonary function, and reduced RVA and pulmonary arterial hypertension in this animal model of elastase-induced emphysema.

Respiratory and Systemic Effects of LASSBio596 Plus Surfactant in Experimental Acute Respiratory Distress Syndrome.

Background/Aims: Exogenous surfactant has been proposed as adjunctive therapy for acute respiratory distress syndrome (ARDS), but it is inactivated by different factors present in the alveolar space. We hypothesized that co-administration of LASSBio596, a molecule with significant anti-inflammatory properties, and exogenous surfactant could reduce lung inflammation, thus enabling the surfactant to reduce edema and improve lung function, in experimental ARDS. Methods: ARDS was induced by cecal ligation and puncture surgery in BALB/c mice. A sham-operated group was used as control (CTRL). After surgery (6 hours), CTRL and ARDS animals were assigned to receive: (1) sterile saline solution; (2) LASSBio596; (3) exogenous surfactant or (4) LASSBio596 plus exogenous surfactant (n = 22/group). Results: Regardless of exogenous surfactant administration, LASSBio596 improved survival rate and reduced collagen fiber content, total number of cells and neutrophils in PLF and blood, cell apoptosis, protein content in BALF, and urea and creatinine levels. LASSBio596 plus surfactant yielded all of the aforementioned beneficial effects, as well as increased BALF lipid content and reduced surface tension. Conclusion: LASSBio596 exhibited major anti-inflammatory and anti-fibrogenic effects in experimental sepsis-induced ARDS. Its association with surfactant may provide further advantages, potentially by reducing surface tension.

Ghrelin therapy improves lung and cardiovascular function in experimental emphysema

BackgroundEmphysema is a progressive disease characterized by irreversible airspace enlargement followed by a decline in lung function. It also causes extrapulmonary effects, such as loss of body mass and cor pulmonale, which are associated with shorter survival and worse clinical outcomes. Ghrelin, a growth-hormone secretagogue, stimulates muscle anabolism, has anti-inflammatory effects, promotes vasodilation, and improves cardiac performance. Therefore, we hypothesized that ghrelin might reduce lung inflammation and remodelling as well as improve lung mechanics and cardiac function in experimental emphysema.MethodsForty female C57BL/6 mice were randomly assigned into two main groups: control (C) and emphysema (ELA). In the ELA group (n=20), animals received four intratracheal instillations of pancreatic porcine elastase (PPE) at 1-week intervals. C animals (n=20) received saline alone (50 μL) using the same protocol. Two weeks after the last instillation of saline or PPE, C and ELA animals received ghrelin or saline (n=10/group) intraperitoneally (i.p.) daily, during 3 weeks. Dual-energy X-ray absorptiometry (DEXA), echocardiography, lung mechanics, histology, and molecular biology were analysed.ResultsIn elastase-induced emphysema, ghrelin treatment decreased alveolar hyperinflation and mean linear intercept, neutrophil infiltration, and collagen fibre content in the alveolar septa and pulmonary vessel wall; increased elastic fibre content; reduced M1-macrophage populations and increased M2 polarization; decreased levels of keratinocyte-derived chemokine (KC, a mouse analogue of interleukin-8), tumour necrosis factor-α, and transforming growth factor-β, but increased interleukin-10 in lung tissue; augmented static lung elastance; reduced arterial pulmonary hypertension and right ventricular hypertrophy on echocardiography; and increased lean mass.ConclusionIn the elastase-induced emphysema model used herein, ghrelin not only reduced lung damage but also improved cardiac function and increased lean mass. These findings should prompt further studies to evaluate ghrelin as a potential therapy for emphysema.

Variability in Tidal Volume Affects Lung and Cardiovascular Function Differentially in a Rat Model of Experimental Emphysema

In experimental elastase-induced emphysema, mechanical ventilation with variable tidal volumes (VT) set to 30% coefficient of variation (CV) may result in more homogenous ventilation distribution, but might also impair right heart function. We hypothesized that a different CV setting could improve both lung and cardiovascular function. Therefore, we investigated the effects of different levels of VT variability on cardiorespiratory function, lung histology, and gene expression of biomarkers associated with inflammation, fibrogenesis, epithelial cell damage, and mechanical cell stress in this emphysema model. Wistar rats (n = 35) received repeated intratracheal instillation of porcine pancreatic elastase to induce emphysema. Seven animals were not ventilated and served as controls (NV). Twenty-eight animals were anesthetized and assigned to mechanical ventilation with a VT CV of 0% (BASELINE). After data collection, animals (n = 7/group) were randomly allocated to VT CVs of 0% (VV0); 15% (VV15); 22.5% (VV22.5); or 30% (VV30). In all groups, mean VT was 6 mL/kg and positive end-expiratory pressure was 3 cmH2O. Respiratory system mechanics and cardiac function (by echocardiography) were assessed continuously for 2 h (END). Lung histology and molecular biology were measured post-mortem. VV22.5 and VV30 decreased respiratory system elastance, while VV15 had no effect. VV0, VV15, and VV22.5, but not VV30, increased pulmonary acceleration time to pulmonary ejection time ratio. VV22.5 decreased the central moment of the mean linear intercept (D2 of Lm) while increasing the homogeneity index (1/β) compared to NV (77 ± 8 μm vs. 152 ± 45 μm; 0.85 ± 0.06 vs. 0.66 ± 0.13, p < 0.05 for both). Compared to NV, VV30 was associated with higher interleukin-6 expression. Cytokine-induced neutrophil chemoattractant-1 expression was higher in all groups, except VV22.5, compared to NV. IL-1β expression was lower in VV22.5 and VV30 compared to VV0. IL-10 expression was higher in VV22.5 than NV. Club cell protein 16 expression was higher in VV22.5 than VV0. SP-D expression was higher in VV30 than NV, while SP-C was higher in VV30 and VV22.5 than VV0. In conclusion, VV22.5 improved respiratory system elastance and homogeneity of airspace enlargement, mitigated inflammation and epithelial cell damage, while avoiding impairment of right cardiac function in experimental elastase-induced emphysema.

Distensibility index of the inferior vena cava in experimental acute respiratory distress syndrome.

We determined the accuracy of distensibility index of inferior vena cava (dIVC) for evaluation of fluid responsiveness in rats with acute respiratory distress syndrome (ARDS) and validated this index for use in rat models. In protocol 1, E. coli lipopolysaccharide was administered in Wistar rats (n=7). After 24h, animals were mechanically ventilated, and stroke volume (SV) and dIVC quantified after blood drainage and subsequent volume expansion (albumin 20%). A receiver operating characteristic (ROC) curve was plotted to determine the optimal dIVC cutoff. In protocol 2, rats (n=10) were divided into fluid-responders (SV increase >5%) and nonresponders (SV increase <5%). The dIVC cutoff obtained from protocol 1 was 25%. Fluid responders had a 2.5 relative risk of low dIVC (<25%). The sensitivity, specificity, positive predictive, and negative predictive values for dIVC were 74%, 62%, 59%, and 76%, respectively. In conclusion, a dIVC threshold <25% was associated with positive response after volume expansion and could be used to titrate fluids in endotoxin-induced ARDS.

Pressure-support improves lung protection and reduces cardiovascular dysfunction compared to pressure-controlled ventilation in experimental emphysema

Acute exacerbations of pulmonary emphysema lead to increased morbidity, mortality and, in some cases, requirement of invasive mechanical ventilation (MV) [1]. So far, no study has compared the biological impact of pressure-controlled ventilation (PCV) versus pressure-support ventilation (PSV) in experimental emphysema. Our aims were to develop an elastase-induced emphysema model in rats, and to compare the effects of PCV and PSV on the lung and heart.