(A) Left graph, Macrophages from Chop+/+;Apoe−/− or Chop−/−;Apoe−/− mice were loaded with lipoprotein-derived unesterified cholesterol and then assayed for apoptosis. *, P < 0.01. Middle graph, Macrophages were incubated for 18 h with vehicle control (0.2% ethanol) or 50 μM 7-ketocholesterol and then assayed for apoptosis. *, P<0.01 compared with Chop+/+;Apoe−/− macrophages. Right graph, Macrophages were incubated for 15 h with vehicle control, 50 μg/ml oxidized LDL (OxLDL), 1 mM SIN-1, or both compounds. The cells were then assayed for apoptosis. Asterisk = P<0.01 compared with Chop+/+;Apoe−/− macrophages. In the case of cholesterol loading, ~5% of apoptotic cells were PI-positive. With 7-ketocholesterol and OxLDL + SIN-1, the percentage of PI-positive cells was >50%, indicating advanced apoptosis, and both annexin-positive and PI-positive cells were decreased in the CHOP–deficient macrophages.
(B) Efferocytosis assays were conducted using various combinations of peritoneal macrophages from Chop+/+;Apoe−/− and Chop−/−;Apoe−/− mice, where the macrophages served as the source of either the apoptotic cells or the efferocytes. None of the differences shown are statistically significant.
(C) Representative micrographs show less TUNEL-positive signal (red; arrows) in nuclei (blue) of aortic root lesions from Chop−/−;Apoe−/− lesions. Bar, 10 μm. Quantification of TUNEL-positive nuclei was conducted on lesions from 20 Chop+/+;Apoe−/− mice and 21 Chop−/−;Apoe−/− mice. *, P<0.01.
(D) Representative micrographs show less activated caspase-3 (red; arrows) in aortic root lesions from Chop−/−;Apoe−/− vs. Chop−/−;Apoe−/− lesions. Bar, 10 μm. Quantification of TUNEL-positive cells was conducted on lesions from 7 Chop+/+;Apoe−/− mice and 7 Chop−/−;Apoe−/− mice. *, P<0.05.
(E) Representative sequential sections of a Chop+/+;Apoe−/− lesion stained for nuclei (Hoechst), TUNEL, and macrophages. Bar, 10 μm.