Triggering role of acid sphingomyelinase in endothelial lysosome-membrane fusion and dysfunction in coronary arteries

Abstract

The present study determined whether activation of acid sphingomyelinase (ASM) drives membrane proximal lysosomes to fuse to the cell surface, facilitating membrane lipid rafts (LRs) clustering in coronary arterial endothelial cells (CAECs) and leading to endothelial dysfunction. By confocal microscopy, the activators of ASM, phosphatidylinositol (PI), and bis (monoacylglyceryl) phosphate (Bis), and an inducer of ASM, butyrate, were found to increase LRs clustering in bovine CAECs, which was blocked by lysosome fusion inhibitor vacuolin-1. However, arsenic trioxide (Ars), an inducer of de novo synthesis of ceramide, had no such effect. Similarly, vacuolin-1-blockable effects were observed using fluorescence resonance energy transfer detection. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis demonstrated that all of these treatments, even Ars, increased ceramide production in CAECs. When ASM gene was silenced, all treatments except Ars no longer increased ceramide levels. Furthermore, dynamic fluorescence monitoring in live cells showed that PI and Bis stimulated lysosome-membrane fusion in CAECs. Functionally, PI and Bis impaired endothelium-dependent vasodilation in perfused coronary arteries, which was blocked by vacuolin-1 and a lysosome function inhibitor, bafilomycine. FasL (Fas ligand), a previously confirmed lysosome fusion stimulator as a comparison, also produced a similar effect. It is concluded that ASM activation serves as a triggering mechanism and driving force, leading to fusion of membrane proximal lysosomes into LR clusters on the cell membrane of CAECs, which represents a novel mechanism mediating endothelial dysfunction during death receptor activation or other pathological situation.

Ebolavirus Requires Acid Sphingomyelinase Activity and Plasma Membrane Sphingomyelin for Infection

“…Finally, we showed that EBOV infection is completely blocked by vacuolin-1, a specific inhibitor of lysosomal exocytosis and ASMase translocation to the plasma membrane (4,8). This drug was specific for EBOV GP activity, as infection by VSV-G pseudotyped virus was unaffected after treatment.…”

“…ASMase appears to be recruited to sites of VLP binding or may induce binding of VLPs to cells. ASMase can be brought to the cell surface by an exocytosis-related mechanism (4,51). This may also bring other endosome-localized proteins to the cell surface.…”

“…Then, SM is either recycled back to the plasma membrane via exocytosis or delivered to lysosomes, where it is hydrolyzed to ceramide and phosphocholine by ASMase (31). However, membrane damage and the binding of microbial pathogens can result in the translocation of lysosomal ASMase to the outer leaflet of the plasma membrane, where it cleaves surface-exposed SM (4,51). The conversion of the SM in rafts to ceramide can result in raft enlargement, receptor clustering, membrane invagination, and macropinosome formation (22)(23)(24)59), all of which promote the uptake of particles, including viruses, into cells.…”

Lipid Raft Redox Signaling: Molecular Mechanisms in Health and Disease

“…Given that NOX activation requires many cofactors to work together, LRs provide a wonderful platform, for NOX and the other NADPH oxidase subunits and cofactors, to assemble and then work as an active enzymatic complex. Indeed, many studies, in house and outside, have demonstrated that LRs even provide the driving force that promotes the assembling of NOX with other NADPH oxidase subunits or cofactors (26,27,190,235,388,389,391,443,444,446,452,453).…”

“…This leads to the activation of NADPH oxidase and production of O 2 -, resulting in prominent amplification of the transmembrane signals. Besides lipid components such as SM, ceramide and cholesterol, many other molecules, such as NADPH oxidase subunits, Rac, and other regulatory components are clustered to form complex signalosomes (27,190,335,446 (76,106,121,333).…”