The selective PGI2 receptor agonist selexipag ameliorates Sugen 5416/hypoxia-induced pulmonary arterial hypertension in rats

Fig 1. Hemodynamic effects of selexipag in U46619-induced acute PAH model rats. Effects of selexipag on right ventricular systolic pressure (RVSP) (A), mean arterial blood pressure (MAP) (B) and heart rate (HR) (C). The percentage change in RVSP, MAP and HR from the value at 0 min were calculated for each measurement until 120 min. Statistical analyses were performed using repeated measures ANOVA followed by Student’s t-test. **P<0.01 vs. vehicle. Values are means ± S.E.M. N = 8 per group.

Fig 2. Study design using the SuHx rat model. Scheme of the early-stage study (A) and the late-stage study (B). Black arrowheads indicate the date for the assessment of the outcome parameters.

Fig 3. Hemodynamic effects of selexipag in the early stage of SuHx rats. Effect of selexipag on right ventricular systolic pressure (RVSP) (A), the ratio of the weight of the right ventricle to that of the left ventricle + septum (RV/LV+S) (B), mean arterial blood pressure (MAP) (C) and heart rate (HR) (D). Statistical analyses were performed using Tukey’s test. ##P<0.01 vs. normoxia, ††P<0.01 vs. SuHx-3w, *P<0.05 and **P<0.01 vs. SuHx-6w. Values are means ± S.E.M. N = 8–10 per group.

Fig 4. Effect of selexipag on occlusive lesions in the early stage of SuHx rats. Representative photographs of occlusive lesions of pulmonary vessels (scale bars = 20 μm) (A) and the percentage of occlusive lesions among the vessels (B). Statistical analysis was performed using Tukey’s test. ##P<0.01 vs. normoxia, ††P<0.01 vs. SuHx-3w, **P<0.01 vs. SuHx-6w. Values are means ± S.E.M. N = 8–10 per group.

Fig 5. Hemodynamic effects of selexipag in the late stage of SuHx rats. RVSP (A), RV/LV+S (B), mean arterial blood pressure (MAP) (C) and heart rate (HR) (D). Statistical analyses were performed using Tukey’s test. ##P<0.01 vs. normoxia, ††P<0.01 vs. SuHx-5w, **P<0.01 vs. SuHx-8w. Values are means ± S.E.M. N = 10 per group.

Fig 6. Effect of selexipag on occlusive lesions in the late stage of SuHx rats. Representative photographs of occlusive lesions (top; scale bar = 20 μm) and medial wall thickness of pulmonary vessels (bottom; scale bars = 50 μm) (A), the percentage of occlusive lesions among the vessels (B) and the percentage of medial wall thickness (C). Statistical analyses were performed using Tukey’s test. ##P<0.01 vs. normoxia, ††P<0.01 vs. SuHx-5w, **P<0.01 vs. SuHx-8w. Values are means ± S.E.M. N = 10 per group.

Fig 7. Effect of selexipag on cell proliferation and apoptosis in occlusive lesions in the late stage. Cells positive for the cell proliferation marker Ki-67 and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL), a method for identifying apoptotic cells, were detected immunohistochemically (arrows). Representative photomicrographs of vessels from each group (top, Ki-67; bottom, von Willebrand factor; scale bars = 20 μm) (A), percentage of Ki-67-positive vessels (B) and representative photomicrographs of vessels with TUNEL-positive cells (C). Statistical analysis was performed using Tukey’s test. ##P<0.01 vs. normoxia, ††P<0.01 vs. SuHx-5w, **P<0.01 vs. SuHx-8w. Values are means ± S.E.M. N = 10 per group.

Fig 8. Effect of selexipag on expression of collagen type I in the late stage. Collagen type I was detected immunohistochemically. Representative photomicrographs of vessels from each group (scale bar = 20 μm) (A) and the number of positive pixels per square micrometer of vessel area (B). Statistical analysis was performed using Tukey’s test. ##P<0.01 vs. normoxia, ††P<0.01 vs. SuHx-5w, **P<0.01 vs. SuHx-8w. Values are means ± S.E.M. N = 10 per group.

Fig 9. Effect of selexipag on mortality and RV hypertrophy in Fischer rats with SuHx-induced PAH. Kaplan-Meier survival curves for the two groups (A) and RV/LV+S in the surviving animals on day 42 (B). Statistical analysis was performed using log-rank analysis for survival and Student’s t-test for RV/LV+S. *P<0.05 vs. vehicle. N = 10 per group.