Anti-atherogenic capabilities of Angptl 4 in vivo. ApoE?/?Angptl 4+/+ and ApoE?/?Angptl 4?/? male mice were fed a normal rodent chow diet (Clea, Tokyo, Japan). Whole
apexbio were collected and stained with Sudan IV, and 6-μm-thick frozen cross sections of the aortic sinus were prepared and stained with oil red O according to the method described by SRL Inc. (Tokyo, Japan) [13]. The size of the atherosclerotic area was measured by BZ-II analysis software (Keyence, Osaka, Japan). Experiments using cultured macrophages. Peritoneal macrophages were collected and cultured from male mice as previously described [14]. Lipoprotein preparation and modification of native LDL. Human LDL (d = 1.019–1.063 g/ml) and Oxidized-LDL was prepared as previously described [14]. Acetyl-LDL (acLDL) was prepared by chemical modification of LDL with acetic anhydride as previously described [15]. The peritoneal macrophages were incubated for 5 h at 37 °C with 50 μg/mL acLDL [13]. Foam cell formation (CE-accumulation) assay. The peritoneal macrophages were incubated with 50 μg/mL acLDL for 24 h in the presence of 0.1 mmol/L [3H]oleate conjugated with BSA, and cellular
lipids were extracted to determine the radioactivity of cholesteryl-[3H]oleate as described previously [13].
