Histological analysis

After completion of right heart catheterization, all animals were euthanized, and both the heart and lungs were dissected and frozen in liquid nitrogen or fixed in 4% paraformaldehyde for further analysis. To evaluate vascular remodelling, the lung was perfused with 4 °C DPBS and perfusion-fixed with 10% formaldehyde solution for 24 h. The whole lung was embedded in paraffin, and sections were made and stained with haematoxylin and eosin (H&E). H&E staining was used to determine the pulmonary artery media thickness (PAMT). At least 10 images of pulmonary arterioles (diameter between 30 and 100 μm) per rat were captured and analysed. The PAMT is defined as the distance between the inner and outer elastic lamina. The vessel external diameter (ED) of the pulmonary arteriole was determined. The relative PAMT (%) was calculated as 100 × 2PAMT/ED.

Fixed lung tissues were stained with anti-alpha-smooth muscle actin (α-SMA, 1:200, Abcam, USA) to detect pulmonary vascular muscularization using immunohistochemistry. Image-Pro Plus version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to assess the integrated optical density (IOD) value of the α-SMA IHC section. IODs were obtained as the ratio of sum optical density (OD) to the sum area. Lung tissues were stained with anti-Ki67 (1:200, Abcam, USA) and anti-CD68 (1:100, Abcam, USA) using immunohistochemistry.

The right ventricle (RV) wall was removed from the left ventricle (LV) and the septum (S). The ratio of the weight of RV to the weight of LV + S was calculated to determine the extent of RV hypertrophy.