PECAM-1 protects against endothelial cell apoptosis. (A) Endothelial cell apoptosis in PECAM-1^+/+ versus PECAM-1^-/- following thoracic x-irradiation. The top 2 panels show immunohistochemical evaluation of PECAM-1 expression in pulmonary endothelium. PECAM-1 expression is completely absent from lung tissue in the knock-out animals. The bottom 6 panels show immunohistochemical staining for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) to detect fragmented DNA. TUNEL-positive pulmonary vessel endothelial cells have brown staining nuclei. Magnification, × 500. Positive signal is seen as a brown precipitate confined to the nucleus. (B) Quantitative evaluation of TUNEL-positive vessel endothelial cells. Percentage of apoptotic endothelial cells as determined by TUNEL positivity in 15 to 20 randomly selected large pulmonary vessels was evaluated from frozen lung sections at 0, 24, and 48 hours following x-ray irradiation of the thorax. Analysis of variance (ANOVA) yielded a P value of less than .001. (C) Aortic endothelial cells (AECs) derived from wild-type or PECAM-1—deficient C57BL/6 mice were grown in microtiter wells for up to 5 passages. Cell extracts were prepared from one set of wells, normalized for protein content, and their caspase activity determined as described in “Materials and methods.” Following addition of staurosporine, PECAM-1—deficient murine AECs reached near-maximal levels of cytosolic caspase activity nearly 8 hours earlier than did wild-type cells and were nearly all dead within 1 day. Data shown are the mean ± standard deviation of duplicate determination of a single representative experiment of 3 such performed. (D) Phase and fluorescent microscopic analysis of cell death. Nuclear morphology was assessed at the indicated time points using Hoechst dye 33358. A low-power (× 10) view (left panels) of untreated cells shows characteristic endothelial cell cobblestone morphology, while × 40 views of cellular nuclei show initially healthy chromatin, with progressive nuclear condensation with time following addition of staurosporine. This was especially pronounced in PECAM-1^-/- AECs. PECAM-1^+/+ cells appear much more firmly attached 4 hours after staurosporine treatment than do AECs lacking PECAM-1 (middle panels). Lower insets show magnified (approximately × 100, digitally) representative Hoechst-stained nuclei of AECs at various stages of condensation from PECAM-1—deficient AECs (taken from circled nuclei in the panel directly above).