Fig 1.
Progression of pulmonary hypertension and an occlusive pulmonary vasculopathy in Sugen/hypoxia rats.
Effects of Sugen/hypoxia (SuHx) or hypoxia on body weight (A), right ventricular systolic pressure (RVSP) (B), the weight ratio of the right ventricle to the left ventricle + septum (RV/LV+S) (C), percentage of vessels accompanied by occlusive lesions among the small pulmonary arteries per lung section (D), and percentage of vessels accompanied by cellular intimal thickening or intimal fibrosis in all the small pulmonary arteries per lung section (E) during the experimental period. Body weight, RVSP, and R/VL+S ratio among 3 study groups at each time point (A, B, C) or these hemodynamic and morphological parameters at various time points (B, C, D, E) were compared with a one-way analysis of variance followed by Tukey-Kramer multiple comparison test. P<.05 vs. control; †P<.05 vs. hypoxia. F: Correlation between the percentage of occlusive lesions and RVSP (Pearson product-moment correlation coefficients). Values are mean ± SD.
Fig 2.
Cellular components in intimal and plexiform lesions.
Photomicrographs (elastic van Gieson staining, EVG) of sprouting intimal lesion (Panels Aab) and sprouting plexiform lesion (Panel Ac) in rats 3 weeks after initial treatment. Serial sections of longitudinal views (Panels Ba-h) and cross sectional views (Panels Bi-l) of intimal lesions and serial sections of plexiform lesions (Panels Bm-p) in EVG, von Willebrand factor (VWF), α-smooth muscle actin staining (αSMA) and negative controls. Photomicrographs (confocal microscopy) of a section of the intimal and plexiform lesion (Panels C), by using antibodies for VWF and αSMA, were presented. TO-PRO-3, nuclear staining. Arrows indicate abnormal cells.
Fig 3.
Myofibroblasts in plexiform lesions.
Photomicrographs of serial sections of complex plexiform lesions in a rat 13 weeks after initial treatment (Panels A) and sprouting plexiform lesions (Panels B, C, D). Immunohistochemical findings using antibodies for various antibodies were presented. αSMA+, vimentin+ supporting cells, which underlies endothelial monolayers, were regarded as myofibroblasts and were negative for SM1 or SM2. PCNA indicates proliferating cell nuclear antigen. An arrow head indicates fragments of elastic laminae. Abbreviations are described in Fig. 2.
Fig 4.
Immature smooth muscle cells in cellular intimal lesions.
Photomicrographs of serial sections of cellular intimal lesions (Panels A, B). Cells staining positive for αSMA, SM1 and HHF35 but weakly positive or negative with SM2 and CGA7 were regarded as representing phenotypically modulated immature smooth muscle cells. Arrows indicate immature smooth muscle cells. Other abbreviations are described in Figs. 2 and 3.
Fig 5.
Mature smooth muscle cells in intimal fibrosis.
Photomicrographs of serial sections of intimal fibrosis. Cells staining positive for αSMA, SM1, SM2, HHF35 and CGA7 were regarded as representing mature smooth muscle cells. Arrows indicate mature smooth muscle cells. Abbreviations are described in Figs. 2 and 3.
Fig 6.
Quantitative analysis of the proportion of mature and immature smooth muscle cell-dominant lesion in the intimal lesions.
The lesions in which immature smooth muscle cell is the predominant phenotype of cells (> 50%) accounted for 87.5% (n = 21) in lesions with cellular intimal thickening and 14.0% (n = 4) in lesions with intimal fibrosis (chi-square test, p<.0001). Forty-nine intimal lesions, in which histological classification of the intima and immunophenotyping of smooth muscle cells were performed in serial sections, from 20 rats (n = 4 at 3 week, 4 at 5 week, 6 at 8 week, and 6 at 13 week) were evaluated.
Fig 7.
Inflammatory cells in Sugen/hypoxia rats.
A: Photomicrographs of CD68-positive macrophages and pulmonary vascular lesions in a Sugen/hypoxia rat 3 weeks after initial treatment. B: Number of perivascular CD68-positive macrophages per vessel in control, hypoxia, and Sugen/hypoxia groups at 3 and 5 weeks was compared with a one-way analysis of variance followed by Tukey-Kramer multiple comparison test. Number of perivascular CD68-positive macrophages per vessel (C) and the percentage of macrophage-positive intima (D) at different time points were compared with a one-way analysis of variance followed by Tukey-Kramer multiple comparison test. Values are mean ± SD. E: Positive correlation between the number of perivascular macrophages per vessel in a lung section and the percentage of occlusive lesions in a lung section (Pearson product-moment correlation coefficients). F: Percentage of the number of perivascular macrophages per vessel in that of total perivascular inflammatory cells, including macrophages, CD3+ T cells, and mast cells, at different time points were compared with a one-way analysis of variance followed by Tukey-Kramer multiple comparison test.
Fig 8.
Time course of PAH-related inflammatory gene expression in Sugen/hypoxia rats.
Messenger RNA level of various genes at different time points and in controls was compared with a one-way analysis of variance followed by Tukey-Kramer multiple comparison test. Open square (control) indicates the control group 3 weeks after the vehicle treatment; closed square indicates Sugen/hypoxia rats at respective time points. IL6, interleukin 6; MCP1, monocyte chemotactic protein 1; MMP9, matrix metalloproteinase 9; TIMP1, tissue inhibitor of metalloproteinase 1; RANTES, Regulated on Activation, Normal T Cell Expressed and Secreted. Data are expressed as fold-change compared with the control group. Values are mean ± SD.
Fig 9.
Differential expression of PAH-related inflammatory genes in Sugen/hypoxia rats.
Messenger RNA expression level of IL6, MCP1, MMP9, TIMP1, and cathepsin S was compared among Sugen/hypoxia, hypoxia, and control rats at 3 or 5 weeks after initial treatment, with a one-way analysis of variance followed by Tukey-Kramer multiple comparison test. Data are expressed as fold-change compared with the control group. Values are mean ± SD. Abbreviations were described in Fig. 8.
Fig 10.
Summary: Phenotypically modulated SMCs and inflammation in the progression of obstructive pulmonary vasculopathy.