Metabolomics analysis of the lung.

<p>SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 5 weeks (8-wks time point) or 32 weeks (35-wks time point). Metabolomics analysis was performed in the lung tissues. Volcano plots (on the left) and Partial least squares Discriminant Analysis (PLS-DA; on the right) for (A) 8-weeks PAH (green circles) vs. normal control (red circles), (B) 8-weeks PAH (green circles) vs. 35-weeks PAH (red circles) and (C) 35-weeks PAH (green circles) vs. normal control (red circles) are shown. Dot volcano plots represent fold changes (FC) on the x-axis and the statistical significance in <i>P</i> value on the y-axis. Pink dots represent values with <i>P</i>≤0.05 and changes of greater than twofold.</p

Myofibroblasts in the RV.

<p>SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 14 or 32 weeks (17- and 35-wks time points, respectively). Hearts were fixed in formalin and embedded in paraffin. Longitudinal sections of the RV myocardium were subjected to immunohistochemistry using the antibody against (A) α-smooth muscle actin and (B) periostin. Results are presented at x200 and x1,000 magnifications. Scale bars indicate 200 μm for x200 and 50 μm for x1,000. The brown stains indicate the expression of α-smooth muscle actin or periostin. Arrows depict that neither α-smooth muscle actin nor periostin stains are present in fibroblasts of the normal RV; brown α-smooth muscle actin and periostin stains are present overlapping nuclear stains of fibroblasts at 17-wks; and α-smooth muscle actin and periostin stains in fibroblasts are absent at 35-wks.</p

RV hemodynamics.

<p>SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 2, 5, 14 or 32 weeks (5-, 8-, 17- and 35-wks time points, respectively). Animals were anaesthetized, ventilated and the chest opened. A Millar catheter was inserted into the RV apex and hemodynamic measurements were performed. (A) Flow diagram of the experiments. (B) Representative hemodynamic traces. (C) Means ± SEM of RVSP. (D) Means ± SEM of dP/dt_max. (E) Means ± SEM of the ratio of RV weight to LV + septum weight. *Significantly different from each other at <i>P</i> < 0.05. **Significantly different from each other at <i>P</i> < 0.01. ***Significantly different from each other at <i>P</i> < 0.001.</p

RV damage and fibrosis.

<p>SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 2, 5, 14 or 32 weeks (5-, 8-, 17- and 35-wks time points, respectively). (A) Representative H&E staining results of the RV tissue sections. (B) Masson’s trichrome stain results in the RVs. Blue stains indicate fibrotic areas. Bar graph represents means ± SEM of % fibrotic area (N = 7). *Significantly different from each other at <i>P</i> < 0.05. **Significantly different from each other at <i>P</i> < 0.01. (C) Western blotting showing COLA1 expression as an indicator of fibrosis in the RVs at 17 and 35 weeks after the SU5416 injection. Bar graph represents means ± SEM of the ratio of COLA1 to G3PDH. *Significantly different from the control value.</p

Lung structure.

<p>SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 14 or 32 weeks (17- and 35-wks time points, respectively). The large panels on the left are representative VVG stains at x100 magnification. Sections of different sizes of pulmonary arteries (50-150nm) are enlarged (x1000) on the right, showing VVG stain, Masson’s trichrome (MT) stain and immunohistochemistry of α-smooth muscle actin (αSMA).</p