Figure 5

Transplantation of EC clones promotes reversible occlusive pulmonary arteriopathy and severe PH in rats exposed to chronic hypoxia. (A) Representative images of immunohistochemistry (IHC) for vWF and α-SMA (brown staining) demonstrating occlusive pulmonary artery remodelling in hypoxic rats that received EC clones (occlusion with vWF⁺ cells, arrows), but not in CD117⁻ EC transplanted rats (upper panel). Images show overview image obtained at 100× magnification and high-power images of representative pulmonary arteries (400× or 600× magnification). For the “chronic hypoxia 21 days + CD117⁺ EC clones” group, examples of occlusive vascular lesions are shown in high-power images at 600× magnification. In the low-power images, arrows indicate pulmonary arteries exhibiting occlusive lesions. In the high-power images, black arrowheads indicate multiple vascular channels within a pulmonary arterial lesion, and the white arrowheads indicate vWF⁺ cells occluding a pulmonary artery. Note the occlusion is cellular as demonstrated by haematoxylin⁺ cell nuclei (arrowheads). Some occluding cells were also α-SMA⁺(white arrows, lower panel). 21 days after cessation of cHx, the vascular changes were almost completely resolved (lower panel), except for elevated media wall thickness (MWT). Scale bars: 100 μm (low power), 25 μm (high power). (B) RVSP and (C) Fulton index. (D) MWT expressed as fraction of external diameter (ED). (E) Fraction of completely occluded pulmonary arteries. Scatter plots show single data points, mean and SEM. n = 3 (hypoxia, hypoxia+CD117⁻ EC), n = 6 (EC clones). *P < 0.05, ***P < 0.001. (F) Representative Western blot demonstrates time course of caspase 3 cleavage in the lung tissue of rats treated with EC clones, followed by 21 days of chronic hypoxia (cHx), 21 days of cHx and 7 days of normoxia (Nx), and 21 days of cHx and 21 days of Nx. The data indicate a progressive increase in the expression of cleaved caspase-3 in the lung tissue of EC clones + cHx rats after cessation of cHx. β-actin was used as loading control.