Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D.
Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D.
复制标题
高级糖化终产物通过抑制组织蛋白酶 D 抑制人脐静脉内皮细胞的增殖
DOI：
10.3390/ijms18020436
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发表时间：
2017-02-17
影响因子：
5.6
通讯作者：
Sun Y
中科院分区：
文献类型：
--
作者：
Li Y;Chang Y;Ye N;Dai D;Chen Y;Zhang N;Sun G;Sun Y
我们旨在研究晚期糖基化终产物（AGEs）对人脐静脉内皮细胞（HUVECs）增殖和迁移能力的影响。采用甲基噻唑四氮唑（MTT）法、实时细胞分析仪和5-乙基-2′-脱氧尿苷（EdU）染色检测细胞增殖情况。采用创面愈合法和transwell法检测细胞迁移。AGEs显著抑制HUVECs的增殖和迁移，并呈时间和剂量依赖性。Western blotting结果显示，AGEs显著增加了微管相关蛋白1轻链3 (LC3) II/I和p62的表达。p62的免疫荧光和吖啶橙染色显示，AGEs分别显著增加了p62的表达和自噬空泡的积累。氯喹（Chloroquine， CQ）可进一步促进LC3 II/I和p62的表达，增加自噬空泡的积累，促进AGEs诱导的细胞损伤。此外，AGEs以时间依赖性的方式降低组织蛋白酶D （CTSD）的表达。过表达野生型CTSD可显著降低lc3ii /I的比值以及AGEs诱导的p62积累，而过表达无催化活性突变型CTSD则无此影响。只有过表达野生型CTSD才能恢复被AGEs抑制的HUVECs的增殖。然而，野生型CTSD和催化失活突变型CTSD的过表达都能促进被AGEs抑制的HUVECs的迁移。综上所述，我们的研究发现，AGEs抑制HUVECs的增殖和迁移，促进自噬通量，从而对AGEs诱导的细胞损伤起保护作用。由于CTSD具有催化活性，它可以独立于自噬-溶酶体途径促进ages处理的HUVECs的增殖。同时，无论其酶活性如何，CTSD都能促进ages处理的HUVECs的迁移。
We aimed to investigate the effect of advanced glycation end products (AGEs) on the proliferation and migration ability of human umbilical vein endothelial cells (HUVECs). Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay, real-time cell analyzer and 5-Ethynyl-2′-deoxyuridine (EdU) staining. Cell migration was detected by wound-healing and transwell assay. AGEs significantly inhibited the proliferation and migration of HUVECs in a time-and dose-dependent way. Western blotting revealed that AGEs dramatically increased the expression of microtubule-associated protein 1 light chain 3 (LC3) II/I and p62. Immunofluorescence of p62 and acridine orange staining revealed that AGEs significantly increased the expression of p62 and the accumulation of autophagic vacuoles, respectively. Chloroquine (CQ) could further promote the expression of LC3 II/I and p62, increase the accumulation of autophagic vacuoles and promote cell injury induced by AGEs. In addition, AGEs reduced cathepsin D (CTSD) expression in a time-dependent way. Overexpression of wild-type CTSD significantly decreased the ratio of LC 3 II/I as well as p62 accumulation induced by AGEs, but overexpression of catalytically inactive mutant CTSD had no such effects. Only overexpression of wild-type CTSD could restore the proliferation of HUVECs inhibited by AGEs. However, overexpression of both wild-type CTSD and catalytically inactive mutant CTSD could promote the migration of HUVECs inhibited by AGEs. Collectively, our study found that AGEs inhibited the proliferation and migration in HUVECs and promoted autophagic flux, which in turn played a protective role against AGEs-induced cell injury. CTSD, in need of its catalytic activity, may promote proliferation in AGEs-treated HUVECs independent of the autophagy-lysosome pathway. Meanwhile, CTSD could improve the migration of AGEs-treated HUVECs regardless of its enzymatic activity.