Phosphatidylethanolamine Induces an Antifibrotic Phenotype in Normal Human Lung Fibroblasts and Ameliorates Bleomycin-Induced Lung Fibrosis in Mice

Luis Vazquez-De-Lara,José Cisneros-Lira,Mario Garcia-Carrasco,Yair Romero,Fernando Vazquez-De-Lara,Roberto Berra-Romani,René De-La-Rosa Paredes,Francesco Moccia,Beatriz Tlatelpa-Romero,Jaime M Justo-Janeiro,Nora Fernández-Tamayo,Criselda Mendoza-Milla

doi:10.3390/ijms19092758

Abstract

Lung surfactant is a complex mixture of phospholipids and specific proteins but its role in the pathogenesis of interstitial lung diseases is not established. Herein, we analyzed the effects of three representative phospholipid components, that is, dipalmitoilphosphatidylcoline (DPPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), on collagen expression, apoptosis and Ca2+ signaling in normal human lung fibroblasts (NHLF) and probed their effect in an experimental model of lung fibrosis. Collagen expression was measured with RT-PCR, apoptosis was measured by using either the APOPercentage assay kit (Biocolor Ltd., Northern Ireland, UK) or the Caspase-Glo 3/7 assay (Promega, Madison, WI, USA) and Ca2+ signaling by conventional epifluorescence imaging. The effect in vivo was tested in bleomycin-induced lung fibrosis in mice. DPPC and PG did not affect collagen expression, which was downregulated by PE. Furthermore, PE promoted apoptosis and induced a dose-dependent Ca2+ signal. PE-induced Ca2+ signal and apoptosis were both blocked by phospholipase C, endoplasmic reticulum pump and store-operated Ca2+ entry inhibition. PE-induced decrease in collagen expression was attenuated by blocking phospholipase C. Finally, surfactant enriched with PE and PE itself attenuated bleomycin-induced lung fibrosis and decreased the soluble collagen concentration in mice lungs. This study demonstrates that PE strongly contributes to the surfactant-induced inhibition of collagen expression in NHLF through a Ca2+ signal and that early administration of Beractant enriched with PE diminishes lung fibrosis in vivo.

Highlights

A common pathological feature in acute lung injury and diverse interstitial lung diseases, such as idiopathic pulmonary fibrosis (IPF), is the migration of fibroblasts and myofibroblasts from the interstitium to the alveolar spaces [1,2,3]
In other study [8], we found that Beractant promoted apoptosis and reduced the expression levels of type I collagen through an increase in intracellular Ca2+ concentration ([Ca2+]i) that was initiated by the recruitment of phospholipase C (PLC)
To elucidate which components of surfactant participate in this process, we evaluated the effect of three representative phospholipids that are found in normal pulmonary surfactant: DPPG (PC class), PG and PE

Summary

Introduction

A common pathological feature in acute lung injury and diverse interstitial lung diseases, such as idiopathic pulmonary fibrosis (IPF), is the migration of fibroblasts and myofibroblasts from the interstitium to the alveolar spaces [1,2,3]. In the process of migration through partially disrupted and denuded epithelial basement membranes, fibroblasts are exposed to the components of alveolar spaces including surfactant lipids and proteins. We showed that pulmonary surfactant promotes programmed cell death of normal human lung fibroblasts and induces the upregulation of matrix metalloproteinase-1 and the downregulation of type I collagen [7]. We used Beractant, a natural bovine lung extract containing phospholipids, neutral lipids, fatty acids and surfactant-associated hydrophobic proteins B and C. Neither DPPC nor PG were able to induce an increase in [Ca2+]i, which suggests that other surfactant components induce apoptosis and regulate gene expression in normal lung fibroblasts (NHLF)

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