Department of Pharmacology and Toxicology, School of Medicine, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, VA, 23298, USA.
J Mol Med (Berl). 2014 May;92(5):473-85. doi: 10.1007/s00109-014-1120-y. Epub 2014 Jan 25.
Recent studies have indicated a protective role of autophagy in regulating vascular smooth muscle cells homeostasis in atherogenesis, but the mechanisms controlling autophagy, particularly autophagy maturation, are poorly understood. Here, we investigated whether acid sphingomyelinase (ASM)-regulated lysosome function is involved in autophagy maturation in coronary arterial smooth muscle cells (CASMCs) in the pathogenesis of atherosclerosis. In coronary arterial wall of ASM-deficient (Smpd1⁻/⁻) mice on Western diet, there were high expression levels of both LC3B, a robust marker of autophagosomes (APs), and p62, a selective autophagy substrate, compared with those in wild-type (Smpd1⁺/⁺) mice. By Western blotting and flow cytometry, atherogenic stimulation of Smpd1⁺/⁺ CASMCs with 7-ketocholesterol was found to significantly enhance LC3B expression and increase the content of both APs and autophagolysosomes (APLs). In Smpd1⁻/⁻ CASMCs, such 7-ketocholesterol-induced increases in LC3B and p62 expression and APs were further augmented, but APLs formation was abolished. Analysis of fluorescence resonance energy transfer between fluorescence-labeled LC3B and Lamp1 (lysosome marker) showed that 7-ketocholesterol markedly induced fusion of APs with lysosomes in Smpd1⁺/⁺ CASMCs, which was abolished in Smpd1⁻/⁻ CASMCs. Moreover, 7-ketocholesterol-induced expression of cell dedifferentiation marker vimentin and proliferation was enhanced in Smpd1⁻/⁻ CASMCs compared with those in Smpd1⁺/⁺ CASMCs. Lastly, overexpression of ASM further increased APLs formation in Smpd1⁺/⁺ CASMCs and restored APLs formation in Smpd1⁻/⁻ CASMCs indicating that increased ASM expression is highly correlated with enhanced APLs formation. Taken together, our data suggest that the control of lysosome trafficking and fusion by ASM is essential to a normal autophagic flux in CASMCs, which implicates that the deficiency of ASM-mediated regulation of autophagy maturation may result in imbalance of arterial smooth muscle cell homeostasis and thus serve as an important atherogenic mechanism in coronary arteries.
Acid sphingomyelinase (ASM) controls autophagy maturation in smooth muscle cells. ASM maintains smooth muscle cell homeostasis and its contractile phenotype. ASM plays a protective role in smooth muscle dysfunction and atherosclerosis.
最近的研究表明自噬在动脉粥样硬化发生过程中调节血管平滑肌细胞稳态方面具有保护作用，但控制自噬的机制，特别是自噬成熟的机制，尚不清楚。在这里，我们研究了酸性鞘磷脂酶（ASM）调节的溶酶体功能是否参与动脉粥样硬化发病过程中冠状动脉平滑肌细胞（CASMC）中的自噬成熟。在西方饮食下 ASM 缺陷（Smpd1⁻/⁻）小鼠的冠状动脉壁中，LC3B 的表达水平较高，LC3B 是自噬体（APs）的一个强有力的标志物，而 p62 是一种选择性自噬底物，与野生型（Smpd1⁺/⁺）小鼠相比。通过 Western 印迹和流式细胞术，发现致动脉粥样硬化刺激 Smpd1⁺/⁺ CASMC 用 7-酮胆固醇可显著增强 LC3B 的表达，并增加 APs 和自噬溶酶体（APLs）的含量。在 Smpd1⁻/⁻ CASMC 中，这种 7-酮胆固醇诱导的 LC3B 和 p62 表达以及 APs 的增加进一步增强，但 APLs 的形成被消除。荧光共振能量转移分析荧光标记的 LC3B 和 Lamp1（溶酶体标记）之间的荧光共振能量转移显示，7-酮胆固醇显著诱导 Smpd1⁺/⁺ CASMC 中 APs 与溶酶体融合，而在 Smpd1⁻/⁻ CASMC 中则被消除。此外，与 Smpd1⁺/⁺ CASMC 相比，Smpd1⁻/⁻ CASMC 中细胞去分化标志物波形蛋白和增殖的表达增强。最后，ASM 的过表达进一步增加了 Smpd1⁺/⁺ CASMC 中 APLs 的形成，并恢复了 Smpd1⁻/⁻ CASMC 中 APLs 的形成，表明增加的 ASM 表达与增强的 APLs 形成高度相关。综上所述，我们的数据表明，ASM 对溶酶体运输和融合的控制对于 CASMC 中的正常自噬通量至关重要，这表明 ASM 介导的自噬成熟调节的缺失可能导致动脉平滑肌细胞稳态失衡，从而成为冠状动脉中的一个重要动脉粥样硬化机制。
酸性鞘磷脂酶（ASM）控制平滑肌细胞中的自噬成熟。ASM 维持平滑肌细胞的稳态和收缩表型。ASM 在平滑肌功能障碍和动脉粥样硬化中起保护作用。
