Abstract 12584: Transcription Factor EB Regulates Vascular Smooth Muscle Calcification
Abstract 12584: Transcription Factor EB Regulates Vascular Smooth Muscle Calcification
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摘要 12584：转录因子 EB 调节血管平滑肌钙化
DOI：
10.1161/circ.142.suppl_3.12584
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发表时间：
2020
期刊：
影响因子：
37.8
通讯作者：
Morimoto Yuji
中科院分区：
文献类型：
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作者：
Ishiwata Ryo;Morimoto Yuji
简介：血管钙化是心血管相关死亡率的强预测因子。高磷血症引起血管平滑肌细胞（VSMC）的表型转换，导致中膜钙化。最近的研究表明，自噬-溶酶体途径的功能障碍是血管钙化的原因之一，但这一途径失败的过程是难以捉摸的。转录因子EB（TFEB）是溶酶体生物合成的主要调节因子;其在VSMCs中的功能尚不清楚。假设：我们评估了TFEB在VSMCs中的功能障碍是血管钙化的原因的假设。方法和结果：我们在野生型小鼠主动脉中诱导血管钙化，采用无ex体内高磷酸盐血症模型。添加1.7 mmol/L的无机磷酸盐5天可降低TFEB蛋白表达（0.23 ± 0.10倍vs. day 0，n= 5-7）。免疫组化和茜素红染色显示，图尼卡中膜中TFEB表达的减少与钙化的形成相关。从大鼠主动脉分离VSMCs，体外培养7天。无机磷酸盐的添加剂量依赖性地降低了全细胞裂解物和核部分中的TFEB蛋白表达（分别为对照的0.07± 0.03倍，n= 5 ;对照的0.01 ± 0.003倍，n= 4），而它反而增加了Tfeb的mRNA表达（4.48 ± 0.95倍，n= 7，对照）。无机磷酸盐引起的TFEB蛋白质的减少与TFEB在SDS不溶性组分中的积累相关，表明形成了蛋白质聚集体。通过siRNA敲低VSMC中的TFEB加剧了磷酸盐诱导的钙沉积（2.93 ± 0.85倍，n= 4对比阴性siRNA）。通过LysoTracker染料测定，无机磷酸盐的加入显著降低了溶酶体活性;用20 nmol/L的巴弗洛霉素A（一种溶酶体抑制剂）处理进一步加速磷酸盐诱导的钙沉积（13.6 ± 3.69倍，n= 7 vs. vehicle）。功能性TFEB的减少倾向于血管钙化，可能是通过下调溶酶体活性。
Introduction:Vascular calcification is a strong predictor of cardiovascular-related mortality. Hyperphosphatemia causes phenotypic switches of vascular smooth muscle cells (VSMCs), leading to medial calcification. Recent studies have suggested that dysfunction of the autophagy-lysosomal pathway in VSMCs is a cause of vascular calcification, but the process through which this pathway fails is elusive. Transcription factor EB (TFEB) is a master regulator of lysosome biogenesis; its function in VSMCs is unknown.Hypothesis:We assessed the hypothesis that the dysfunction of TFEB in VSMCs is a cause of vascular calcification.Methods and Results:We induced vascular calcification in wild type mouse aorta with anex vivohyperphosphatemia model.Addition of inorganic phosphate at a 1.7 mmol/L for five days decreased TFEB protein expression (0.23 ± 0.10-fold vs. day0,n= 5-7). Immunohistochemistry and alizarin red staining showed that a decrease in TFEB expression in the tunica media was correlated with the formation of calcification. VSMCs were isolated from rat aorta and were cultured for seven daysin vitro. Addition of inorganic phosphate dose-dependently decreased TFEB protein expression both in whole cell lysate and in nuclear fraction (0.07± 0.03-fold vs. control,n= 5 ; 0.01 ± 0.003-fold vs. control,n= 4, respectively) while it rather increased mRNA expression ofTfeb(4.48 ± 0.95-fold,n= 7 vs. control). The Decrease in TFEB protein by inorganic phosphate was correlated with the accumulation of TFEB in the SDS-insoluble fraction, suggesting the formation of protein aggregates. The Knockdown of TFEB in VSMCs by siRNA exacerbated phosphate-induced calcium deposition (2.93 ± 0.85-fold,n= 4 vs. negative siRNA). The addition of inorganic phosphate significantly decreased lysosomal activity determined by LysoTracker dye; and treatment with 20 nmol/L of bafilomycin A, a lysosome inhibitor, further accelerated phosphate-induced calcium deposition (13.6 ± 3.69-fold,n= 7 vs. vehicle).Conclusion:In conclusion, TFEB expression in VSMCs is downregulated at the protein level by hyperphosphatemia. The diminution of functional TFEB predisposes to vascular calcification, presumably through downregulating lysosomal activity.