Figure 1

Effect of sera on intracellular ROS levels and collagen promoter activity. (A-B) Effects of SSc sera on human pulmonary artery smooth muscle cells (HPASMCs) intracellular ROS production. Before stimulation, sub-confluent HPASMCs were loaded with 10 μM of H2-DCFDA and then cultured in basal medium containing 10% (V/V) of sera from scleroderma (SSc) patients with pulmonary arterial hypertension (PAH), without PAH (No PAH) and healthy donors (HD) ^††. Variations in intracellular ROS levels were kinetically determined in a 4 hour time-course experiment (Figure 1A) and values at 2 hours (steady state) used for comparison (Figure 1B). Fluorescence data were normalized for protein content and expressed as Relative Fluorescence Units (RFU). (D-E) Effects of SSc sera on HPASMCs collagen (COL1A1) promoter activation. Sub-confluent 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 PAH, no PAH and HD subjects. Variations of COL1A1 promoter activation were kinetically followed for 10 hours (Figure 1D) and values at 8 hours (steady state) used for comparison (Figure 1E). Data are normalized for transduction efficiency by reporting the ratio of COL1A1-LV-tGFP to EF1α-LV-FP602 Relative Fluorescence Units (RFU). (C-F) In selected experiments, HPASMCs were incubated for 1 hour with 5 μM NADPH oxidase specific inhibitor NOX2ds-tat (NOX) before treatment with SSc sera. ^††Healthy donors were matched for gender, race and smoking status. Horizontal lines indicate the median with interquartile range. Kruskall–Wallis one-way analysis of variance followed by post-hoc Dunn’s test for multiple comparisons were used to detect differences among studied groups in Figures 1B and E. Wilcoxon matched-pairs signed rank test was used to determine meaningful differences between pre- and post-NOX treatment pairs in Figures C and F. All statistical analysis were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, San Diego, CA) and p-values <0.05 were considered to be statistically significant.