Fig 2: NTproBNP, IL-6 and sRAGE plasma concentrations are elevated in patients with pulmonary arterial hypertension versus controls. (A,B) NTproBNP plasma concentrations of patients with idiopathic pulmonary arterial hypertension (IPAH, n = 74) and connective tissue disease-associated pulmonary arterial hypertension (CTD-PAH, n = 37) versus controls (CON, n = 29). (C,D) IL-6 plasma concentrations of IPAH (n = 66) and CTD-PAH (n = 35) patients versus controls (n = 27). (E,F) sRAGE plasma concentrations of IPAH (n = 74) and CTD-PAH (n = 37) patients versus controls (n = 29). The scatter plots on the left show the mean ± SEM, the box and whisker plots on the right show the median with interquartile range ± 10–90 percentile. Statistical test: Kruskal–Wallis test, corrected for multiple testing by Dunn’s test. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: IL-6, Interleukin-6; NTproBNP, N-terminal pro-brain natriuretic peptide; sRAGE, soluble receptor for advanced glycation end products.

Fig 3: RAGE mRNA and protein expression, and immunohistochemistry in human lung tissues from end-stage IPAH patients versus controls. (A) The relative RAGE mRNA expression (AGER) normalized to GAPDH was significantly increased in end-stage IPAH patients (n = 7) vs. controls (CON; n = 9). (B) RAGE protein expression was elevated in whole human lung tissues of end-stage IPAH patients (n = 5) vs. controls (n = 4) measured by Western blot, but this difference did not reach statistical significance. Exposure time: 3.0 s. Values are presented as mean ± SEM. Statistical test: unpaired t-test with Welch’s correction, Mann–Whitney U. * p < 0.05. (C) Pulmonary artery of a female lung transplant donor lung (H&E staining; scale bar, 100 µm). (D) Obliterated pulmonary artery of an end-stage 42-year-old female IPAH patient (H&E staining; scale bar, 100 µm). (E) RAGE staining in a lung transplant donor lung of a female donor (scale bar, 100 µm). (F) The representative image of RAGE staining in a 42-year-old female IPAH patient shows the boosted RAGE expression in the intima and media (endothelial cells, smooth muscle cells, fibroblasts) of an obliterated distal pulmonary artery (concentric hypertrophic lesion). Heightened RAGE expression is also evident in perivascular cells in the outer adventitia, likely representing infiltrating proinflammatory cells, such as macrophages and lymphocytes (scale bar, 100 µm).

Fig 4: NTproBNP, IL-6 and sRAGE plasma concentrations in patients with idiopathic pulmonary arterial hypertension (IPAH) and connective tissue disease-associated pulmonary arterial hypertension (CTD-PAH) increase with pulmonary hypertension severity. The World Health Organization (WHO) functional class (FC) system is used as a surrogate for disease severity. Functional classes I-III include both IPAH and CTD-PAH patients. (A,B) Plasma levels of NTproBNP in controls (n = 29), patients with FC I (n = 21) and FC II and III (n = 90). (C,D) Plasma levels of IL-6 in controls (n = 27), patients with FC I (n = 18) and FC II and III (n = 83). (E,F) Plasma levels of sRAGE in controls (n = 29), patients with FC I (n = 21) and FC II and III (n = 90). The scatter plots on the left show the mean ± SEM, the box and whisker plots on the right show the median with interquartile range ± 10-90 percentile. Statistical test: Kruskal–Wallis test, corrected for multiple testing by Dunn’s test. * p < 0.05, *** p < 0.001. **** p < 0.0001. Abbreviations: IL-6, Interleukin-6; NTproBNP, N-terminal pro-brain natriuretic peptide; sRAGE, soluble receptor for advanced glycation end products.

Fig 5: RAGE mRNA expression and immunohistochemistry in children with end-stage PAH and HPAH (heterozygous BMPR2 mutation). (A) The relative RAGE mRNA expression in children transplanted for idiopathic PAH or pulmonary veno-occlusive disease (IPAH + PVOD = PAH, n = 6) vs. children with heritable PAH (HPAH, BMPR2 +/− mutation, n = 4) shows that a heterozygous BMPR2 mutation does not influence the relative RAGE mRNA expression in whole lung tissues from patients undergoing lung transplantation. Values are presented as mean ± SEM. Statistical test: Mann–Whitney U. (B,C) H&E images of distal pulmonary arteries of an 11-year-old patient with PVOD (B) and a 5-year-old patient with BMPR2 +/− mutation (HPAH; C; scale bar, 100 µm). (D,E). The corresponding images of RAGE staining show increased RAGE expression in vascular and inflammatory cells in the intima, media and adventitia of pulmonary arteries in both the 11-year-old patient with PVOD (D) and the 5-year-old patient with BMPR2 +/− mutation (E; scale bar, 100 µm). Abbreviations: BMPR2, bone morphogenetic protein receptor 2; PAH, pulmonary arterial hypertension.