Abstract 513: Control of Lysosome Trafficking and Autophagic Flux by Acid Sphingomyelinase in Coronary Arterial Myocytes: Protective Role in Atherosclerosis
Autophagy occurs in a variety of mammalian cell via several stages of cellular activity including induction, autophagosome (AP) formation, and autophagic flux (i.e. docking and fusion of APs with lysosomes and breakdown of autophagic vesicles). Despite extensive studies on APs formation, so far little is known about the molecular mechanisms regulating autophgic flux. Since ceramide, a hydrolysis product of sphingomyelin by lysosomal acid sphingomyelinase (Asm), is implicated in the control of lysosome function in coronary arterial myocytes (CAMs). The present study tested the hypothesis that Asm is critical for AP fusion with lysosomes in CAMs and thereby protects mice from atherosclerosis. By Western blotting and flow cytometry, proatherogenic stimulation of wild type (Asm+/+) CAMs with 7-ketocholesterol (7K) was found to significantly enhance LC3B expression by 4.6 folds and increase AP positive cells from 6.7% to 20%. Such increases in LC3B expression and AP positive cells were augmented in CAMs with Asm siRNA or CAMs from Asm gene deficient mice (Asm-/-). After overexpression of GFP-LC3B (AP marker) and RFP-Lamp1 (lysosome marker), 7K-induced fusion of APs with lysosomes, an indicator for the formation of autophagolysosomes (APLs) was abolished in Asm-/- CAMs compared to Asm+/+ CAMs, which was further confirmed by flow cytometry of late autophagy using acridine orange. Consistently, an autophagy degrading protein, p62, was accumulated upon proatherogenic stimulation in Asm-/- CAMs, but not in Asm+/+ CAMs. In whole animal experiments, Asm-/- mice but not Asm+/+ mice on the Western diet exhibited atherosclerotic phenotype, namely, arterial wall thickening, lipid deposition, CAM dedifferentiation and extracellular matrix deposition. In coronary arterial wall of Asm-/- mice, there was tremendous APs accumulation and p62 aggregation and inclusion in the muscle layer. These data together indicate that the control of lysosome trafficking and fusion by Asm or its product is essential to a normal autophagic flux in CAMs and that the deficiency of Asm control of autophagic flux may be an important pathogenic mechanism of atherogenesis in coronary arteries.
- © 2012 by American Heart Association, Inc.