Significance of nuclear upregulation of LOXL2 in fibroblasts and myofibroblasts in the fibrotic process of acute respiratory distress syndrome

Background: Fibrotic scarring is an important prognostic factor for acute respiratory distress syndrome (ARDS). Lysyl oxidase-like 2 (LOXL2), an amine oxidase, is thought to make an integral contribution to fibrotic scarring by facilitating collagen cross-linking. LOXL2 has also been proposed to epigenetically regulate gene expression to direct cell fate. Recent clinical outcomes demonstrated the ineffectiveness of an inhibitory monoclonal antibody against LOXL2 in patients with organ fibrosis, including fibrosis of the lungs, suggesting that sole targeting of the extracellular activity of LOXL2 is insufficient to prevent fibrotic scarring. Moreover, the involvement of LOXL2 in human ARDS remains unknown. Methods: Expression dynamics of LOXL2 during the pathological process of bleomycin (BLM)-induced lung injury model were measured by qRT-PCR and western blotting. The principal cell types expressing LOXL2 and its cellular localization were determined by immunohistochemistry and confocal microscopy. Mlg 2908, a murine lung fibroblast line, was transfected with LOXL2 siRNA or control siRNA, and then assessed for transforming growth factor (TGF)-β1-induced Axin2+ myofibrogenic progenitor (AMP) marker expression, actin filament expression, myofibroblast differentiation, collagen expression, and alteration of the TGF-β1/Smad/Snail pathway by phalloidin staining, qRT-PCR, western blotting, and immunocytochemistry. Levels of LOXL2 in bronchoalveolar lavage fluid (BALF) in patients with ARDS and control subjects were measured by enzyme-linked immunosorbent assay. Results: LOXL2 expression was elevated in lung tissue in the fibrotic phase after injury. Nuclear upregulation of LOXL2 in fibroblasts and myofibroblasts, as well as nuclear upregulation of Snail in myofibroblasts, was evident in fibrotic lung tissue after injury. LOXL2 knockdown blocked TGF-β1-induced AMP marker expression, appearance of proto-myofibroblasts, evolution of differentiated myofibroblasts, collagen expression, and pSmad2/Snail induction in lung fibroblasts. We detected high levels of LOXL2 protein in the BALF from patients with ARDS, specifically during the fibrotic phase. Conclusions: Our results revealed that nuclear LOXL2 is upregulated in lung fibroblasts and myofibroblasts after injury and is required for generation of myofibroblast and maintenance of AMP. The high levels of LOXL2 in human ARDS lung suggest that inhibition of both intracellular and extracellular LOXL2 activity represents an attractive therapeutic target for ARDS and lung scarring.