Fig. 3.

Development of transgenic model for deficiency of HIF1-α and HIF2-α within vascular endothelium. A: schematic illustrating the combination of four individual transgenic mice resulting in the desired model. B: Xgal staining in a lung section from an adult VECad.Cre.HIF1-α^fl/fl.HIF2-α^fl/fl.ROSA.Stop.lacZ (HIF1/2 KO) mouse. Endothelial cells from the large vessels down to the capillaries (lung parenchyma) stain blue. ×20 magnification. C: confocal microscopic images on isolated pulmonary microvascular endothelial cells (PMVECs) from the gene-targeted mice [VECad.Cre positive (HIF1/2 KO)] demonstrated dual immunofluorescence for the endothelial cell marker vWF and the reporter system-based β-galactosidase (β-gal), a pattern not seen in PMVECs from the littermate controls [VECad.Cre negative (Control)]. D: immunohistochemical staining for HIF1-α and HIF2-α in vehicle (PBS) and bleomycin-exposed mice. Vascular endothelial cells were immunostained positive for HIF1-α and HIF2-α at 3 wk after bleomycin administration in control mice (arrowheads), but not in HIF1/2 KO mice. Scale bars 50 μm at ×60 magnification. Counterstain with methyl green. E: quantitative PCR of PMVEC lysates for HIF1-α and HIF2-α, under hypoxic conditions (1% O₂ for 6 h), demonstrating a marked reduction in expression in the HIF-targeted mice. n = 3 per group, *P < 0.05. F: Western blot for HIF1-α from these same PMVECs demonstrated 40% increased blotting intensity of HIF1-α in hypoxia in control cells compared with HIF1/2 KO cells. Immunoblot for HIF1-α is shown with β-actin for loading control.