No upregulation of lectin-like oxidized low-density lipoprotein receptor-1 in serum-deprived EA.hy926 endothelial cells under oxLDL exposure, but increase in autophagy.
The oxidized low-density lipoprotein (oxLDL)-dependent activation of the lectin-like oxLDL receptor-1 (LOX-1) triggers apoptosis in vascular cells and appears to be involved in atherosclerosis. Autophagy might be an alternate to apoptosis in endothelial cells. The EA.hy926 endothelial cell line has been reported to undergo necrosis under oxLDL stimulation. For this reason, we studied the expression of LOX-1 and its oxLDL-dependent function in EA.hy926 cells under serum starvation. Untreated and oxLDL-treated cells expressed the LOX-1 protein at similar levels 6h after starvation. After 24h without oxLDL and with native LDL (nLDL), statistically significant higher levels were found in LOX-1 than in the oxLDL-treated probes. The oxLDL cultures with low LOX-1 expression displayed stronger features of autophagy than those with nLDL as there were remodelling of actin filaments, disrupture of adherens junctions (immunofluorescence staining), and autophagosomes with the characteristic double membrane at the ultrastructural level. For the advanced oxLDL exposure times (18 and 24 h), autophagic vacuoles/autophagolysosomes were morphologically identified accompanied by a decrease in lysosomes. The autophagosome marker protein MAP LC3-II (Western blotting) was significantly augmented 6 and 18 h after oxLDL treatment compared with cultures treated with nLDL and medium alone. Signs of apoptosis were undetectable in cultures under oxLDL exposure, yet present under staurosporin (apoptosis inducer), i.e. presence of apoptotic bodies and cleaved caspase 3. We conclude that serum starvation upregulates LOX-1 in EA.hy926 cells, whereas the additional oxLDL treatment downregulates the receptor and intensifies autophagy probably by increase in oxidative stress.
Nowicki
Marcin
M
Institute of Anatomy, University of Leipzig, Liebigstrasse 13, D-04103 Leipzig, Germany.
Zabirnyk
Olga
O
Duerrschmidt
Nicole
N
Borlak
Jürgen
J
Spanel-Borowski
Katharina
K
D008841:Actin Cytoskeleton / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D008247:Lysosomes / Q000378:metabolism; D018384:Oxidative Stress; D010587:Phagocytosis; D010588:Phagosomes; D051127:Scavenger Receptors, Class E / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism; D015854:Up-Regulation
D015820:Cadherins; D016895:Culture Media, Serum-Free; D008077:Lipoproteins, LDL; C495137:OLR1 protein, human; D051127:Scavenger Receptors, Class E; C099252:oxidized low density lipoprotein; D019311:Staurosporine