Antioxidant Activity Mediates Pirfenidone Antifibrotic Effects in Human Pulmonary Vascular Smooth Muscle Cells Exposed to Sera of Idiopathic Pulmonary Fibrosis Patients

Figure 1

(a-b) Real-time assessment of intracellular ROS production and collagen I synthesis in HPASMCs exposed to sera of IPF patients. (a) Before stimulation, subconfluent human pulmonary artery smooth muscle cells (HPASMCs) were loaded with 10 μM of H₂-DCFDA and then cultured in basal medium containing 10% (v/v) of sera from idiopathic pulmonary fibrosis (IPF), sera from idiopathic pulmonary fibrosis patients treated for 24 weeks with Pirfenidone (IPF + D), and healthy donors (HD). Variations in intracellular ROS levels were kinetically determined in a 5-hour time-course (a randomly selected representative experiment is reported) and values at 2 hours (steady state) were used in the future comparisons. Fluorescence data were normalized for protein content and expressed as relative fluorescence units (RFUs). (b) Before stimulation, subconfluent HPASMCs were transduced with lentiviral particles obtained from the COL1A1-LV-tGFP and EF1α-LV-FP602 lentivectors and then cultured in basal medium containing 10% (v/v) of sera from idiopathic pulmonary fibrosis (IPF), sera from idiopathic pulmonary fibrosis patients treated for 24 weeks with Pirfenidone (IPF + D), and healthy donors (HD). Variations of COL1 promoter activation were kinetically followed for 10 hours (a randomly selected representative experiment is reported) and values at 8 hours (steady state) were used in the future comparison. Data are normalized for transduction efficiency by reporting the ratio of COL1A1-LV-tGFP to EF1α-LV-FP602 relative fluorescence units (RFUs).

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