Nitric oxide attenuates epithelial-mesenchymal transition in alveolar epithelial cells

Abstract

Patients with interstitial lung diseases, such as idiopathic pulmonary fibrosis (IPF) and bronchopulmonary dysplasia (BPD), suffer from lung fibrosis secondary to myofibroblast-mediated excessive ECM deposition and destruction of lung architecture. Transforming growth factor (TGF)-␤1 induces epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AEC) to myofibroblasts both in vitro and in vivo. Inhaled nitric oxide (NO) attenuates ECM accumulation, enhances lung growth, and decreases alveolar myofibroblast number in experimental models. We therefore hypothesized that NO attenuates TGF-␤1-induced EMT in cultured AEC. Studies of the capacity for endogenous NO production in AEC revealed that endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) are expressed and active in AEC. Total NOS activity was 1.3 pmol ⅐ mg protein Ϫ1 ⅐ min Ϫ1 with 67% derived from eNOS. TGF-␤1 (50 pM) suppressed eNOS expression by more than 60% and activity by 83% but did not affect iNOS expression or activity. Inhibition of endogenous NOS with L-NAME led to spontaneous EMT, manifested by increased ␣-smooth muscle actin (␣-SMA) expression and a fibroblast-like morphology. Provision of exogenous NO to TGF-␤1-treated AEC decreased stress fiber-associated ␣-SMA expression and decreased collagen I expression by 80%. NO-treated AEC also retained an epithelial morphology and expressed increased lamellar protein, E-cadherin, and pro-surfactant protein B compared with those treated with TGF-␤ alone. These findings indicate that NO serves a critical role in preserving an epithelial phenotype and in attenuating EMT in AEC. NO-mediated regulation of AEC fate may have important implications in the pathophysiology and treatment of diseases such as IPF and BPD. alveolar epithelium; lung injury; nitric oxide synthases; pulmonary fibrosis; transforming growth factor-␤ IDIOPATHIC PULMONARY FIBROSIS (IPF), bronchopulmonary dysplasia (BPD), and post-acute respiratory distress syndrome fibrosing alveolitis are interstitial lung diseases that result in significant long-term morbidity and mortality for affected patients (5,9,11,27). Clinical morbidity associated with interstitial lung disease arises from excessive and disordered extracellular matrix (ECM) production, proliferation and activation of pulmonary fibroblasts, destruction of lung architecture, and fibrosis. Once thought to be exclusively secondary to dysregulated inflammation in the lung, recent studies suggest that the ECM accumulation and fibrosis associated with these conditions result instead from chronic epithelial injury and activation independent of inflammation (34).Whereas the normal response to lung injury is migration and proliferation of type II pneumocytes to reestablish an intact epithelial lining (3), the chronic and repetitive injury that culminates in fibrotic lung disease promotes hyperplasia and dysplasia of alveolar epithelial cells (AEC). Injured and activated AEC then participate in aberrant cytokine signaling that perpetuates the fibro...

An Improved Assay for Measuring Low Levels of Nitric Oxide in Cultured Pulmonary Myofibroblasts

“…This expression pattern is consistent with other work in alveolar epithelial cells where the majority of NOS activity is attributable to eNOS [13]. Furthermore, we have also shown that NOS transcription and enzyme activity change as a consequence of cell differentiation.…”

“…It is well established that eNOS associates reversibly with a diverse family of proteins that regulate its trafficking in the cell and such intracellular targeting is a crucial step in regulating its activity ( [31] and references therein). Moreover, the observed differentiation-dependent fluctuations in NO likely implicates this signaling pathway in regulating the proto-myofibroblast to myofibroblast transition since in other cell types NO is known to be an important inhibitor of epithelial-mesenchymal transitions [13].…”

Bronchiolitis obliterans after allogeneic hematopoietic SCT: further insight—new perspectives?

“…Previous studies could highlight NO having a critical role in preserving an epithelial phenotype and in attenuating epithelial-mesenchymal transition in alveolar epithelial cells. 35 Furthermore, several studies have suggested a beneficial impact of NO on pulmonary disorders. [36][37][38] It might be concluded that vice versa a decrease of NO may result in an elevated susceptibility to lung fibrosis.…”

Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1α

“…Indeed, elevated concentrations of exhaled NO and increased epithelial expression of NOS2 are characteristic features of chronic inflammatory airway diseases such as asthma (1,2). The biological roles of NOS2 within the airway epithelium include contribution to innate host defense (3,4), regulation of epithelial ion transport (5), and maintenance of epithelial barrier integrity (1,6,7). In addition, our recent studies showed that physiological concentrations of NO can promote airway epithelial cell migration and repair in response to in vitro injury, which was associated with increased expression and activation of gelatinase B (matrix metalloproteinase-9; MMP-9) (8).…”