Roflumilast N- oxide combined with sildenafil reverses cellular remodeling on IPF models

Background: Idiopathic pulmonary fibrosis (IPF) is characterized by a rapid progressive lung decline and premature death after its diagnosis. Roflumilast displayed anti-fibrotic effects in animal and cellular models. Recent studies indicate that the combination of PDE4 and PDE5 inhibitors (sildenafil) potentiates anti-fibrotic properties of each drug, suggesting potential beneficts of this combination. Objectives: To study the effects from adding sildenafil to roflumilast N-oxide (RNO) inhibiting TGFβ1-induced human alveolar type II (AECII) epithelial-to-mesenchymal transition (EMT) and human fibrocyte to myofibroblast transition in vitro. Methods: AECII and fibrocytes were isolated from healthy donors. Cells were pre-incubated with therapeutical concentrations of RNO (2nM) and/or sildenafil (10nM) and stimulated with TGFβ1 for 72h. Studies on chemotaxis, cell viability (annexin V), different markers of EMT and myofibroblast were performed. Results: RNO/sildenafil combination significantly reduced TGFβ1- induced gene and protein expression of mesenchymal/myofibroblast markers collagen tye I, alpha actin smooth muscle and vimentin, prevented the loss of epitelial markers E-cadherin and ZO-1 and inhibited the change of cell phenotype observed in the EMT. Inhibition of fibrocyte to myofibroblast transition and fibrocyte chemotaxis was also potentiated by RNO/sildenafil combination. Annexin V studies showed that combination improved survival of AECII. RNO/sildenafil combination showed synergic properties. Conclusions: RNO/sildenafil combination show synergic activity, reduces mesenchymal markers expression and reverses EMT, improving anti-fibrotic effects in human AECII and fibrocytes.