Figure 3

Human pulmonary microvascular ECs (HPMVECs) in serum-free medium conditioned by pulmonary arterial fibroblast-like cells (PAFLCs) or myofibroblast-like cells (MFLCs). The phenotypic alteration of HPMVECs was assessed microscopically and by immunofluorescence staining. A) and D), Before incubation in serum-free medium conditioned by PAFLCs and MFLCs; B) and E), At the 2nd passage after incubation in serum-free medium conditioned by PAFLCs; C) and F), At the 2nd passage after incubation in serum-free medium conditioned by MFLCs; A), B) and C), microscopic findings; the magnification was 100×. Scale bar = 100 μm; D), E) and F), Immunofluorescence staining for anti-Factor VIII (green) and anti-α-SMA (red). The blue staining was DAPI. The magnification was 200×. Scale bar = 50 μm. F), Some cells were positive for smooth muscle actin fibers (see inset); HPMVECs = human pulmonary microvascular endothelial cells; MFLCs = myofibroblast like cells; PAFLCs = fibroblast-like cells from control pulmonary arteries. G) Positive cells for anti-von Willebrand factor and anti-α-SM-actin were counted in 3 different fields at a magnification of × 200 in a fluorescence microscope. *P < 0.05 _VS. PAFLCs, n ≥ 3. H) The TGF-β1 protein levels in the conditioned medium were measured by ELISA. There were no significant differences between the serum-free medium conditioned by PAFLCs and MFLCs.