Atherosclerosis develops in the arterial cell wall when monocytes enter into the subendothelial space, differentiate into macrophages and are converted to cholesteryl ester rich foam cells in the presence of oxidized/ modified LDL. Little is known about altered gene expression in macrophage/foam cells. In the Edwards lab, mRNA Differential Display identified human white/murine ABC8 mRNAs that were transcriptionally induced 25- to 100-fold when macrophages were incubated with Ac-LDL, highly oxidized LDL and/or oxysterols. The research proposed in this application will examine both the function of ABC8 in murine RAW 264.7 macrophages and the expression and cellular localization of ABC8 in mouse arterial lesions. In addition, I propose to identify other sterol-induced ABC proteins, including the dimeric partner of ABC8. Multiple approaches will be utilized to achieve these aims. RAW cells will be treated with antisense RNAs to reduce endogenous ABC mRNA levels. Stably transfected RAW cells that express dominant negative ABC8 will also be isolated. The effect of either antisense oligonucleotides or the presence of mutant ABC8 protein on various cell parameters will be tested. These parameters include: i) the effect on sterol efflux, ii) altered susceptibility to the toxic/killing effects of hydroxysterols, and iii) altered regulation of SREBP-responsive genes. In a second aim, the expression of ABC8 mRNA and protein in arterial mouse lesions will be determined using in situ hybridization and immunocytochemistry. Finally, a degenerate PCR technique will be used to identify ABC mRNAs, including the partner for ABC8, expressed in foam cells. In conclusion, the studies proposed in this application are expected to provide critical insights into the function, expression and localization of ABC8/ABC proteins and their role in cellular sterol homeostasis and atherosclerosis.
