Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D

Abstract

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.

Melatonin protects endothelial progenitor cells against AGE-induced apoptosis via autophagy flux stimulation and promotes wound healing in diabetic mice

“…Chen et al 32 reported that AGEs increased the ratio of LC3-II/I but had no obvious effect on p62 protein expression in HUVECs. Li et al 33 demonstrated that AGEs dramatically increased the expression of LC3-II/I and p62 in HUVECs. In our study, we observed a similar phenomenon in EPCs, as AGEs increased the ratio of LC3-II to LC3-I, while p62 was also accumulated, suggesting the impairment of autophagic flux rather than the activation of autophagy.…”

Consensus guidelines for the use and interpretation of angiogenesis assays

“…In a similar manner, the incorporation of 5-bromo-2′-deoxyuridine (BrdU) or EdU (5-ethynyl-2′deoxyuridine) can be measured. BrdU or EdU can be (in)directly detected and subsequently be (semi-) quantified using ELISA, flow cytometry, or immunohistochemistry [ 57 , 58 , 60 ]. The latter two quantification techniques allow one to determine the fraction of dividing cells.…”

“…For example, the indirect detection of antigens (e.g., PCNA or BrdU) requires careful procedural optimization. Although the alternative “click” chemistry, by which the analogous EdU can be detected directly, circumvents this issue of indirect detection, making EdU preferable [ 57 , 58 , 60 ]. Nonetheless, assay readout and interpretation are important to consider.…”

Antitumor effects of arsenic disulfide on the viability, migratory ability, apoptosis and autophagy of breast cancer cells

“…When autophagy is induced, LC3 is converted from the cytoplasmic form LC3-I into the membrane-associated form LC3-II. The ratio of LC3-II/LC3-I is widely considered as a primary marker of autophagy activation ( 40 , 41 ).…”