Conserved effects of APOE on Cox-2 expression in human and mouse SMCs and mice. (a) Quiescent mouse aortic SMCs were treated with 10% FBS in the absence or presence of 50 μg/ml HDL, 50 μg/ml LDL, 2 μM APOE or 2 μM APOA-I. Changes in the levels of Cox-1, Cox-2, and GAPDH (loading control) mRNAs were determined by RT-PCR and analyzed using NIH Image 1.63 software. (b) Cell lysates made from serum-starved aortic SMCs stimulated with 10% FBS in the absence and presence of 2 μM APOA-I or APOE were immunoblotted using anti–Cox-2 and anti-cdk4 (loading control). Human aortic SMCs (HSMC) were grown to near confluence in 150-mm dishes, serum starved for 48 hours in defined medium, and then directly stimulated with 10% FBS in the absence or presence of 2 μM APOE or 2 μM APOA-I. Changes in the levels of Cox-2 mRNA (c) and protein (d) were determined as in a and b, respectively. Cell lysates were also made from serum-starved human aortic endothelial cells (HAEC) stimulated with 2 μM APOA-I or APOE, and the levels of Cox-2 protein were determined by immunoblotting (d). (e) C57BL/6 mice were injected with adenoviral constructs expressing APOA-I, APOE, or null vector. Total RNA was isolated from the aortae and reverse transcribed into cDNA. Changes in the levels of Cox-1, Cox-2, and GAPDH mRNA were determined as outlined in Methods.