Fig 1.
Hemodynamics characteristics of the OVA/Sugen5416 model of PAH.
(A) Schematics of the rat model of PAH induced by OVA and Sugen5416. Rats were injected intraperitoneally on Days 1 and 8 with 1 mg of OVA with Imject Alum Adjuvant (OVA/Alum) for sensitization. On Day 15, rats were subjected to 30 min of 1% OVA inhalation with a nebulizer, which continued twice a week for four weeks. Sugen5416 (20 mg/kg body weight) was injected subcutaneously (S.C.) once a week on the day after OVA inhalation. Hemodynamic measurements were performed, rats were sacrificed, and lung and heart tissues were obtained on Day 50. (B) Representative traces of hemodynamic measurements taken from the RV using a Millar catheter. (C) The bar graph represents means ± SEM of systolic RV pressure. * denotes a value that is significantly different from the control value at P < 0.05 (N = 6).
Fig 2.
Histological characterizations of the lungs of rats treated with OVA/Sugen5416.
Rats were subjected to treatment with OVA and Sugen5416 to promote PAH. Paraffin-embedded lung sections were subjected to (A—C) H&E stain, (D—F) immunohistochemistry with the α-smooth muscle actin antibody, (G–I) Verhoeff-van Gieson stain and (J—L) Masson’s trichrome stain.
Fig 3.
Rats were subjected to treatment with OVA and Sugen5416 to promote PAH. (A) Fulton ratio, the ratio of RV mass / (LV mass + septum mass). (B) H&E stain of the heart sections. The bar graph represents the ratio of the cross-sectional areas of RV / (LV + septum). (C) Microscopic observations of the H&E-stained RV. (i & ii) The longitudinal section of a representative control RV under 200x and 400x magnifications. (iii & iv) The longitudinal section of a representative RV from PAH rats under 200x and 400x magnifications. (D) Masson’s trichrome stain was performed on RV sections. Muscle fibers stain red. Collagen stains blue. (E) RV homogenates were subjected to western blotting to detect alpha-1 type I collagen (COL1A1) as an indicator of fibrosis. Bar graphs represent means ± SEM. * denote values significantly different from controls at P < 0.05 (N = 6).
Fig 4.
Metabolomics studies of the RV.
Metabolomics analysis were performed in RV tissues from control rats and rats subjected to OVA/Sugen5416 to promote PAH. (A) PLS-DA plots for positive and negative modes of ionization with a 95% confidence region for OVA/Sugen5416 (green circle; N = 6) vs. control (red circle; N = 4). (B) Volcano plot in positive and negative ionization modes for OVA/Sugen5416 vs. control. Dot plots represent fold changes (FC) on the x-axis and the statistical significance in P value (P) on the y-axis. Pink dots represent values with P ≤ 0.05 and a multiplicative change of greater than twofold. (C) Xanthine, (D) uric acid, (E) oxidized glutathione and (F) α-tocopherol nicotinate levels in control rat RVs and RVs of rats treated with OVA/Sugen5416 (OVA/Su). Bar graphs represent means ± SEM. * denote values that are significantly different from control at P < 0.05.
Fig 5.
Oxidative/nitrosative protein modification in the RVs of rats with PAH.
RV homogenates prepared from control rats (Cont) and rats treated with OVA and Sugen5416 (OVA/Su) to promote PAH were subjected to Western blotting with (A) the glutathione antibody to detect S-glutathionylation, (B) the antibody against 2,4-dinitrophenylhydrazine to detect protein carbonyls, (C) 4-hydroxylnonenal (4-HNE) antibody, (D) malondialdehyde (MDA) antibody, (E) nitrotyrosine antibody and (F) nitrosocysteine antibody. Bar graphs represent means ± SEM. * denote values significantly different from controls at P < 0.05 (N = 6).
Fig 6.
Protein S-nitrosylation in the RVs of rats with PAH.
(A) RV homogenates prepared from control rats (Cont) and rats treated with OVA and Sugen5416 (OVA/Su) to promote PAH were immunoprecipitated with the antibody against S-nitrosocysteine (SNO-Cys). Immunoprecipitated samples were subjected to SDS-PAGE and stained with Coomassie Blue. The band indicated by an arrow, which was consistently higher in OVA/Sugen5416-treated (OVA/Su) animals, was analyzed by mass spectrometry. (B & C) Mass spectrometry identifications of HSP90 and SERCA2 were confirmed by subjecting immunoprecipitated samples to Western blotting. (D & E) Western blotting showing that protein expression levels of HSP90 and SERCA2 are not different between RVs of control rats and those of rats with PAH. Bar graphs represent means ± SEM. * denote values significantly different from controls at P < 0.05 (N = 6). (F) RV homogenates prepared from control rats and rats treated with OVA/Sugen5416 were subjected to Dojindo SulfoBiotics Protein S-Nitrosylation Monitoring assay and subsequently to Western blotting with the HSP90 antibody. SNO x1 depicts HSP90 with one cysteine residue nitrosylated.