Highly Selective Endothelin-1 Receptor A Inhibition Prevents Bleomycin-Induced Pulmonary Inflammation and Fibrosis in Mice.

BACKGROUND: Pulmonary fibrosis is a chronic disease, which progressively leads to respiratory failure and ultimately death. Endothelin-1 (ET-1), a vasoconstrictor secreted by endothelial cells, promotes vasoconstriction by activation of its receptors A and B. OBJECTIVES: We addressed the role of highly selective ET-1 receptor A (ETA) inhibition in the pathogenesis of experimental pulmonary fibrosis by bleomycin (BLM). METHODS: BLM sulfate (2 U/mL) or saline was intratracheally administered to C57/Bl6 mice (4 groups; n = 5-11/group). Pretreatment with the highly selective ETA receptor inhibitor sitaxentan (15 mg/kg/day) was started 1 day prior to BLM injection and continued for the duration of the experiment. Lung mechanics were assessed prior to sacrifice at days 7, 14, and 21 after BLM, followed by procurement of bronchoalveolar lavage fluid (BALF), blood, and lung tissue samples. RESULTS: Time-dependent effects of BLM exposure included decreased static compliance and increased lung elastance, airspace inflammation and microvascular permeability, histological acute lung injury and fibrosis, and lung collagen deposition. Pretreatment with highly selective ETA receptor inhibitor had no adverse effect on control mice but improved lung mechanics and lung injury score in addition to decreasing BALF pleocytosis, protein content, and collagen deposition in BLM-treated mice. Mortality from BLM reached 40% and occurred primarily during the inflammatory stage of the model but was abrogated by sitaxentan pretreatment. CONCLUSIONS: We conclude that in our BLM-induced pulmonary fibrosis model, prophylactic highly selective ETA inhibition improves survival, preserves lung function, attenuates lung injury, and reduces collagen deposition.

Vascular endothelial-cadherin downregulation as a feature of endothelial transdifferentiation in monocrotaline-induced pulmonary hypertension.

Increased pulmonary vascular resistance in pulmonary hypertension (PH) is caused by vasoconstriction and obstruction of small pulmonary arteries by proliferating vascular cells. In analogy to cancer, subsets of proliferating cells may be derived from endothelial cells transitioning into a mesenchymal phenotype. To understand phenotypic shifts transpiring within endothelial cells in PH, we injected rats with alkaloid monocrotaline to induce PH and measured lung tissue levels of endothelial-specific protein and critical differentiation marker vascular endothelial (VE)-cadherin. VE-cadherin expression by immonoblotting declined significantly 24 h and 15 days postinjection to rebound to baseline at 30 days. There was a concomitant increase in transcriptional repressors Snail and Slug, along with a reduction in VE-cadherin mRNA. Mesenchymal markers α-smooth muscle actin and vimentin were upregulated by immunohistochemistry and immunoblotting, and α-smooth muscle actin was colocalized with endothelial marker platelet endothelial cell adhesion molecule-1 by confocal microscopy. Apoptosis was limited in this model, especially in the 24-h time point. In addition, monocrotaline resulted in activation of protein kinase B/Akt, endothelial nitric oxide synthase (eNOS), nuclear factor (NF)-κB, and increased lung tissue nitrotyrosine staining. To understand the etiological relationship between nitrosative stress and VE-cadherin suppression, we incubated cultured rat lung endothelial cells with endothelin-1, a vasoconstrictor and pro-proliferative agent in pulmonary arterial hypertension. This resulted in activation of eNOS, NF-κB, and Akt, in addition to induction of Snail, downregulation of VE-cadherin, and synthesis of vimentin. These effects were blocked by eNOS inhibitor N(ω)-nitro-l-arginine methyl ester. We propose that transcriptional repression of VE-cadherin by nitrosative stress is involved in endothelial-mesenchymal transdifferentiation in experimental PH.

Inhibition of HMGCoA reductase by simvastatin protects mice from injurious mechanical ventilation.

BACKGROUND: Mortality from severe acute respiratory distress syndrome exceeds 40% and there is no available pharmacologic treatment. Mechanical ventilation contributes to lung dysfunction and mortality by causing ventilator-induced lung injury. We explored the utility of simvastatin in a mouse model of severe ventilator-induced lung injury. METHODS: Male C57BL6 mice (n = 7/group) were pretreated with simvastatin or saline and received protective (8 mL/kg) or injurious (25 mL/kg) ventilation for four hours. Three doses of simvastatin (20 mg/kg) or saline were injected intraperitoneally on days -2, -1 and 0 of the experiment. Lung mechanics, (respiratory system elastance, tissue damping and airway resistance), were evaluated by forced oscillation technique, while respiratory system compliance was measured with quasi-static pressure-volume curves. A pathologist blinded to treatment allocation scored hematoxylin-eosin-stained lung sections for the presence of lung injury. Pulmonary endothelial dysfunction was ascertained by bronchoalveolar lavage protein content and lung tissue expression of endothelial junctional protein Vascular Endothelial cadherin by immunoblotting. To assess the inflammatory response in the lung, we determined bronchoalveolar lavage fluid total cell content and neutrophil fraction by microscopy and staining in addition to Matrix-Metalloprotease-9 by ELISA. For the systemic response, we obtained plasma levels of Tumor Necrosis Factor-α, Interleukin-6 and Matrix-Metalloprotease-9 by ELISA. Statistical hypothesis testing was undertaken using one-way analysis of variance and Tukey's post hoc tests. RESULTS: Ventilation with high tidal volume (HVt) resulted in significantly increased lung elastance by 3-fold and decreased lung compliance by 45% compared to low tidal volume (LVt) but simvastatin abrogated lung mechanical alterations of HVt. Histologic lung injury score increased four-fold by HVt but not in simvastatin-pretreated mice. Lavage pleocytosis and neutrophilia were induced by HVt but were significantly attenuated by simvastatin. Microvascular protein permeability increase 20-fold by injurious ventilation but only 4-fold with simvastatin. There was a 3-fold increase in plasma Tumor Necrosis Factor-α, a 7-fold increase in plasma Interleukin-6 and a 20-fold increase in lavage fluid Matrix-Metalloprotease-9 by HVt but simvastatin reduced these levels to control. Lung tissue vascular endothelial cadherin expression was significantly reduced by injurious ventilation but remained preserved by simvastatin. CONCLUSION: High-dose simvastatin prevents experimental hyperinflation lung injury by angioprotective and anti-inflammatory effects.

A role for bronchial epithelial autotaxin in ventilator-induced lung injury.

BACKGROUND: The pathophysiology of acute respiratory distress syndrome (ARDS) may eventually result in heterogeneous lung collapse and edema-flooded airways, predisposing the lung to progressive tissue damage known as ventilator-induced lung injury (VILI). Autotaxin (ATX; ENPP2), the enzyme largely responsible for extracellular lysophosphatidic acid (LPA) production, has been suggested to play a pathogenic role in, among others, pulmonary inflammation and fibrosis. METHODS: C57BL/6 mice were subjected to low and high tidal volume mechanical ventilation using a small animal ventilator: respiratory mechanics were evaluated, and plasma and bronchoalveolar lavage fluid (BALF) samples were obtained. Total protein concentration was determined, and lung histopathology was further performed RESULTS: Injurious ventilation resulted in increased BALF levels of ATX. Genetic deletion of ATX from bronchial epithelial cells attenuated VILI-induced pulmonary edema. CONCLUSION: ATX participates in VILI pathogenesis.

Orotracheal treprostinil administration attenuates bleomycin-induced lung injury, vascular remodeling, and fibrosis in mice.

Pulmonary fibrosis is a progressive disease characterized by disruption of lung architecture and deregulation of the pulmonary function. Prostacyclin, a metabolite of arachidonic acid, is a potential disease mediator since it exerts anti-inflammatory and anti-fibrotic actions. We investigated the effect of treprostinil, a prostacyclin analogue, in bleomycin-induced experimental pulmonary fibrosis. Bleomycin sulfate or saline was administrated intratracheally to mice (n = 9-10/group) at day 0. Orotracheal aspiration of treprostinil or vehicle was administered daily and started 24 h prior to bleomycin challenge. Evaluation of lung pathology was performed in tissue samples and bronchoalveolar lavage fluid collected 7, 14 and 21 days after bleomycin exposure. Lung injury was achieved due to bleomycin exposure at all time points as indicated by impaired lung mechanics, pathologic lung architecture (from day 14), and cellular and protein accumulation in the alveolar space accompanied by a minor decrease in lung tissue VE-cadherin at day 14. Treprostinil preserved lung mechanics, and reduced lung inflammation, fibrosis, and vascular remodeling (day 21); reduced cellularity and protein content of bronchoalveolar lavage fluid were additionally observed with no significant effect on VE-cadherin expression. Bleomycin-induced collagen deposition was attenuated by treprostinil from day 14, while treprostinil involvement in regulating inflammatory processes appears mediated by NF-κB signaling. Overall, prophylactic administration of treprostinil, a stable prostacyclin analogue, maintained lung function, and prevented bleomycin-induced lung injury, and fibrosis, as well as vascular remodeling, a hallmark of pulmonary hypertension. This suggests potential therapeutic efficacy of treprostinil in pulmonary fibrosis and possibly in pulmonary hypertension related to chronic lung diseases.

Differential Expression of Aquaporins in Experimental Models of Acute Lung Injury.

AIM: The mammalian lung expresses at least three aquaporin (AQP) water channels whose precise role in lung injury or inflammation is still controversial. MATERIALS AND METHODS: Three murine models of lung inflammation and corresponding controls were used to evaluate the expression of Aqp1, Aqp4, Aqp5 and Aqp9: lipopolysaccharide (LPS)-induced lung injury; HCl-induced lung injury; and ventilation-induced lung injury (VILI). RESULTS: All models yielded increased lung vascular permeability, and inflammatory cell infiltration in the broncho-alveolar lavage fluid; VILI additionally produced altered lung mechanics. Lung expression of Aqp4 decreased in the models that targeted primarily the alveolar epithelium, i.e. acid aspiration and mechanical ventilation, while Aqp5 expression decreased in the model that appeared to target both the capillary endothelium and alveolar epithelium, i.e. LPS. CONCLUSION: Participation of aquaporins in the acute inflammatory process depends on localization and the type of lung injury.