Arterial Medial Calcification through Enhanced small Extracellular Vesicle Release in Smooth Muscle-Specific Asah1 Gene Knockout Mice
Arterial Medial Calcification through Enhanced small Extracellular Vesicle Release in Smooth Muscle-Specific Asah1 Gene Knockout Mice
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DOI：
10.1038/s41598-020-58568-5
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发表时间：
2020-02-03
影响因子：
4.6
通讯作者：
Li, Pin-Lan
中科院分区：
文献类型：
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作者：
Bhat, Owais M.;Li, Guangbi;Li, Pin-Lan
动脉中膜钙化（AMC）涉及增加的小细胞外囊泡（sEV）分泌和动脉壁中的磷灰石钙沉淀。AMC的介导机制仍然知之甚少。在本研究中，平滑肌特异性酸性神经酰胺酶（Ac）基因敲除小鼠（Asah 1（fl/fl）/SMCre）被用来证明溶酶体神经酰胺信号通路在AMC中的作用。在接受高剂量维生素D后，发现Asah 1（fl/fl）/SMCre小鼠与其同窝小鼠（Asah 1（fl/fl）/SMwt和WT/WT小鼠）相比，主动脉和冠状动脉中的AMC更严重。这些小鼠在动脉中膜中的骨桥蛋白和RUNX 2（成骨标志物）、CD 63、AnX 2（sEV标志物）和ALP表达（矿化标志物）也显著上调。在培养的Asah 1（fl/fl）/SMCre小鼠冠状动脉平滑肌细胞（CASMCs）中，与WT CASMCs相比，高剂量Pi导致钙沉积、表型改变和sEV分泌显著增加，这与溶酶体-多泡体（MVB）相互作用减少有关。此外，GW 4869，sEV释放抑制剂，减少sEV分泌和钙化在这些细胞。GCaMP 3钙离子成像和Port-a-Patch膜片钳技术显示，在Asah 1（fl/fl）/SMCre CASMCs中，调节溶酶体与MVB相互作用的溶酶体瞬时受体电位粘磷脂1（TRPML 1）通道被显著抑制。SMC中的溶酶体Ac通过调节溶酶体TRPML 1通道活性和溶酶体-MVB相互作用来控制sEV释放，这对表型转变和AMC有重要贡献。
Arterial medial calcification (AMC) involves an increased small extracellular vesicle (sEV) secretion and apatite calcium precipitation in the arterial wall. The mechanisms mediating AMC remain poorly understood. In the present study, smooth muscle-specific acid ceramidase (Ac) gene knockout mice (Asah1(fl/fl)/SMCre) were used to demonstrate the role of lysosomal ceramide signaling pathway in AMC. Asah1(fl/fl)/SMCre mice were found to have more severe AMC in both aorta and coronary arteries compared to their littermates (Asah1(fl/fl)/SMwt and WT/WT mice) after receiving a high dose vitamin D. These mice also had pronounced upregulation of osteopontin and RUNX2 (osteogenic markers), CD63, AnX2 (sEV markers) and ALP expression (mineralization marker) in the arterial media. In cultured coronary arterial smooth muscle cells (CASMCs) from Asah1(fl/fl)/SMCre mice, high dose of Pi led to a significantly increased calcium deposition, phenotypic change and sEV secretion compared to WT CASMCs, which was associated with reduced lysosome-multivesicular body (MVB) interaction. Also, GW4869, sEV release inhibitor decreased sEV secretion and calcification in these cells. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with MVBs were found remarkably inhibited in Asah1(fl/fl)/SMCre CASMCs as shown by GCaMP3 Ca2+ imaging and Port-a-Patch patch clamping of lysosomes. Lysosomal Ac in SMCs controls sEV release by regulating lysosomal TRPML1 channel activity and lysosome-MVB interaction, which importantly contributes to phenotypic transition and AMC.