Figure 1. Lung endothelial cells are more susceptible to hyperoxia-induced apoptosis than heart endothelial cells; PKCδ deficiency is protective against lung endothelial cell apoptosis.
Age and sex-matched PKCδ+/+ or PKCδ−/− mice were caged in normoxic (room air) or hyperoxic (~95% O2) environments for 72h. The organs from PKCδ+/+ mice were harvested and immunohistochemically processed either for fragmented DNA, using TUNEL, (panels A and B) or for the identification of apoptotic endothelial cells, using antibodies directed against vWF and counterstained using TUNEL and methyl green for nuclei (panel D). Panels A and B, representative sections of TUNEL stained normoxia and hyperoxia exposed lungs (panel A) and hearts (panel B) are shown at 400X magnification in bright field. Arrows indicate cells with TUNEL staining. Panel C, four random fields from each section viewed at 400X magnification were quantitated for the number of apoptotic cells per field. n = 4; * p < 0.05 vs. normoxic lungs. NS, not significant. Panel D, representative section of a hyperoxic mouse lung section co-stained for vWF (VIP positive; purple staining) and fragmented DNA, using TUNEL (diaminobenzidine (DAB) positive; brown staining) viewed at 1000X magnification in bright field; scale bar = 20µm. Panel E, random fields (16–27 fields) containing apoptotic cells from each section viewed at 1000X magnification were quantitated for the number of TUNEL positive endothelial cells relative to the total number of TUNEL positive cells in the cumulative fields of the PKCδ+/+ organ sections. n = 5. * p < 0.0001. Panel F, the number of apoptotic endothelial cells were quantitated in lung sections from PKCδ+/+ and PKCδ−/− mice exposed to normoxia or hyperoxia co-stained for vWF and fragmented DNA, using TUNEL. n = 3–5. *p < 0.0001.