Abstract
Macrophages use an extracellular, hydrolytic compartment formed by local actin polymerization to digest aggregated LDL (agLDL). Catabolism of agLDL promotes foam cell formation and creates an environment rich in LDL catabolites, including cholesterol and ceramide. Increased ceramide levels are present in lesional LDL, but the effect of ceramide on macrophage proatherogenic processes remains unknown. Here, we show that macrophages accumulate ceramide in atherosclerotic lesions. Using macrophages from sphingosine kinase 2 KO (SK2KO) mice to mimic ceramide-rich conditions of atherosclerotic lesions, we show that SK2KO macrophages display impaired actin polymerization and foam cell formation in response to contact with agLDL. C16-ceramide treatment impaired wild-type but not SK2KO macrophage actin polymerization, confirming that this effect is due to increased ceramide levels. We demonstrate that knockdown of RhoA or inhibition of Rho kinase restores agLDL-induced actin polymerization in SK2KO macrophages. Activation of RhoA in macrophages was sufficient to impair actin polymerization and foam cell formation in response to agLDL. Finally, we establish that during catabolism, macrophages take up ceramide from agLDL, and inhibition of ceramide generation modulates actin polymerization. These findings highlight a critical regulatory pathway by which ceramide impairs actin polymerization through increased RhoA/Rho kinase signaling and regulates foam cell formation.
Highlights
Macrophages use an extracellular, hydrolytic compartment formed by local actin polymerization to digest aggregated LDL
We flushed sterilized femurs and tibias from wild-type (C57BL/6), Sphk1flox/flox Sphk2 / (SK2KO), Sphk1 / Sphk2 / [Sphingosine kinase 1 (SK1)/2KO], S1pr1 / [Sphingosine-1-phosphate 1 (S1P1) KO], and S1pr2 / (S1P2 KO) mice on a C57BL/6 background [as has been described previously [16,17,18]] and from ASM / (A-SMaseKO) and wild-type control [as has been described previously [19]], and cells were differentiated for 7 days by culture in DMEM supplemented with 10% heat-inactivated FBS, 50 U/ml penicillin, and 50 g/ml streptomycin, supplemented with 20% L-929 cell-conditioned media in a humidified atmosphere (5% CO2) at 37°C
We found that lesional macrophages contact ceramide, in regions close to necrotic cores (Fig. 1A, arrows, and Fig. 1C)
Summary
Macrophages use an extracellular, hydrolytic compartment formed by local actin polymerization to digest aggregated LDL (agLDL). We establish that during catabolism, macrophages take up ceramide from agLDL, and inhibition of ceramide generation modulates actin polymerization These findings highlight a critical regulatory pathway by which ceramide impairs actin polymerization through increased RhoA/Rho kinase signaling and regulates foam cell formation.—Singh, R. Exocytosis of lysosomal contents into the LS allows delivery of hydrolytic enzymes, such as lysosomal acid lipase and acid SMase residing in the lysosomes, which promote catabolism of agLDL and generation of catabolites such as free cholesterol [3, 4]. This free cholesterol promotes macrophage actin polymerization, likely through activation of Rac/ Cdc GTPases [3].
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