Abstract 1056: Overexpression Of Lysosomal Acid Lipase

Background: Previous evidence suggests arterial smooth muscle cells (SMCs) comprise the majority of atherosclerotic lesion foam cells in human and mouse atheromas, have reduced expression of ABCA1 and lysosomal acid lipase (LAL) compared to macrophages, and retain cholesteryl esters in lysosomes rather than in the cytoplasm as in macrophage foam cells. Exogenous LAL can increase cholesterol efflux from SMC foam cells despite low ABCA1. Hypothesis: Overexpression of LAL increases cholesterol efflux from SMC foam cells by ABCA1-dependent and -independent mechanisms. Methods: Human vascular SMCs were transfected with 500 MOI AdV- LIPA or AdV- mCherry (adenovirus) control for 6 h and cholesterol loaded using 100 μg/ml aggregated LDL (agLDL) for 24 h. LAL activity was measured by specific enzymatic assay and location of stored cholesteryl esters (CE) was determined using LAMP-1 (lysosome marker) and BODIPY (neutral lipid) staining by confocal microscopy. Cholesterol efflux to 10 μg/ml apoliprotein A-I (ApoA-I) or 100 μg/ml HDL was determined following ABCA1 siRNA treatment or control, with cholesterol mass determined using Amplex Red assay. Student’s t test was used for statistical analysis of LAL activity, and Mann-Whitney U Test for analysis of colocalization of lipid pools. Results: LAL activity increased 1.67-fold 72 h after transfection with AdV- LIPA compared to SMCs transfected with AdV- mCherry (n=5, p = 0.001). Increased LAL expression in the absence of cholesterol acceptor resulted in a shift in CE storage from lysosomes to non-lysosomal compartments (Pearson correlation coefficient for BODIPY/LAMP-1 colocalization 0.27 in AdV- LIPA vs. 0.47 in AdV- mCherry SMCs, n=103, p < 0.00001). Preliminary cholesterol efflux studies indicate SMCs overexpressing LAL release cholesterol by both ABCA1-dependent and -independent mechanisms. Conclusion: Our study provides evidence that despite low ABCA1 and LAL expression, SMC foam cells mobilize lysosomal cholesterol for cellular trafficking and efflux following supplementation with LAL. Further studies will examine changes in gene expression and the mechanism of ABCA1-independent efflux following LAL supplementation in SMC foam cells.