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Chloroquine-Induced Accumulation of Autophagosomes and Lipids in the Endothelium
Authors:Ewelina Bik  Lukasz Mateuszuk  Jagoda Orleanska  Malgorzata Baranska  Stefan Chlopicki  Katarzyna Majzner
Affiliation:1.Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, 14 Bobrzynskiego Str., 30-348 Krakow, Poland; (E.B.); (L.M.); (J.O.); (M.B.); (S.C.);2.Faculty of Chemistry, Jagiellonian University, 2 Gronostajowa Str., 30-387 Krakow, Poland;3.Chair of Pharmacology, Jagiellonian University, 16 Grzegorzecka Str., 31-531 Krakow, Poland
Abstract:Chloroquine (CQ) is an antimalarial drug known to inhibit autophagy flux by impairing autophagosome–lysosome fusion. We hypothesized that autophagy flux altered by CQ has a considerable influence on the lipid composition of endothelial cells. Thus, we investigated endothelial responses induced by CQ on human microvascular endothelial cells (HMEC-1). HMEC-1 cells after CQ exposure were measured using a combined methodology based on label-free Raman and fluorescence imaging. Raman spectroscopy was applied to characterize subtle chemical changes in lipid contents and their distribution in the cells, while the fluorescence staining (LipidTox, LysoTracker and LC3) was used as a reference method. The results showed that CQ was not toxic to endothelial cells and did not result in the endothelial inflammation at concentrations of 1–30 µM. Notwithstanding, it yielded an increased intensity of LipidTox, LysoTracker, and LC3 staining, suggesting changes in the content of neutral lipids, lysosomotropism, and autophagy inhibition, respectively. The CQ-induced endothelial response was associated with lipid accumulation and was characterized by Raman spectroscopy. CQ-induced autophagosome accumulation in the endothelium is featured by a pronounced alteration in the lipid profile, but not in the endothelial inflammation. Raman-based assessment of CQ-induced biochemical changes offers a better understanding of the autophagy mechanism in the endothelial cells.
Keywords:endothelium  Chloroquine  lipids  lysosomotropism  Raman imaging  fluorescence imaging
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