Abstract 628: Overexpression of ABCA1 in Cultured Endothelial Cells Using Helper-Dependent Adenovirus Enhances ApoAI-Mediated Cholesterol Efflux and has Anti-Inflammatory Effects

Background: ABCA1 removes cholesterol from vascular wall cells via apoAI-mediated efflux, generating HDL that transports cholesterol to the liver for excretion. This process of reverse cholesterol transport is atheroprotective; therefore, strategies that increase vascular wall ABCA1 may prevent or reverse atherosclerosis. Cholesterol efflux mediated by ABCA1 and apoAI can also have anti-inflammatory effects; however, excess depletion of cellular cholesterol can cause cell stress and apoptosis. We tested whether transducing endothelial cells (EC) with a helper-dependent adenoviral vector that expresses ABCA1 (HDAdABCA1) enhances apoAI-mediated cholesterol efflux and reduces inflammatory markers without causing cellular toxicity. Methods: We cloned rabbit ABCA1, constructed HDAdABCA1, transduced bovine aortic EC (BAEC) with either HDAdABCA1 or empty vector (HDAdNull). We measured ABCA1 protein by immunoblotting and apoAI-mediated cholesterol efflux by loading EC with 3 [H] cholesterol, then adding apoAI protein to serum-free medium. We assessed EC phenotype using MTT (metabolic activity), BrdU (proliferation), wound-healing assay (migration), and flow cytometry (apoptosis). We measured ICAM-1, VCAM-1, IL-6, and TNFα mRNA in transduced EC by qRT-PCR both under basal conditions and after serum-starvation, addition of apoAI, and LPS challenge. Results: We observed a ~3-fold increase in ABCA1 protein in EC transduced with HDAdABCA1 and a ~2-fold increase in apoAI-mediated cholesterol efflux (P<0.01 for both). This level of ABCA1 overexpression did not alter EC metabolic activity, proliferation, migration, or apoptosis. Under basal conditions, HDAdABCA1 had no effect on inflammatory markers. However, after serum starvation and addition of apoAI, HDAdABCA1-transduced EC had reduced expression of inflammatory markers both before and after LPS treatment. Conclusions: HDAdABCA1 increases EC ABCA1 expression and enhances apoAI-mediated cholesterol efflux, but does not cause toxicity or increase inflammatory markers. In contrast, ABCA1 overexpression appears to have anti-inflammatory effects. Future studies will test if HDAdABCA1 decreases lipid rafts, and whether overexpression of ABCA1 in EC in vivo is atheroprotective.