Endoplasmic reticulum stress is involved in acetylated low-density lipoprotein induced apoptosis in THP-1 differentiated macrophages / 中华医学杂志(英文版)

ABSTRACT

<p><b>BACKGROUND</b>Cardiovascular disease is a major cause of mortality and morbidity in patients with chronic kidney disease. Macrophage death in advanced atherosclerosis promotes necrosis and plaque destabilization. In vitro data from peritoneal macrophages show apoptosis triggered through endoplasmic reticulum (ER) stress caused by free cholesterol accumulation plays an important role. Here we used THP-1 cells differentiated by 100 ng/ml of phorbol 12-myristate 13-acetate (PMA) for five days as an in vitro model, to investigate if acetylated low-density lipoprotein (AcLDL) loading could also induce apoptosis and its underlying mechanisms.</p><p><b>METHODS</b>Oil red O staining was used to examine the lipid droplets. Confocal microscopy was used to visualize the uptake of AcLDL. Hoechst 33258 stain and the caspase 3,7 assay were used to detect apoptosis. High performance liquid chromatography was used in the intracellular free cholesterol (FC) and cholesterol ester (CE) assay. Western blotting was used to demonstrate the protein level. Real-time PCR was used to detect the changes of mRNAs. ER free cholesterol was also assayed.</p><p><b>RESULTS</b>Confocal microscopy showed THP-1 cells differentiated by 100 ng/ml of PMA for five days uptake more AcLDL than differentiated for two days. Hoechst 33258 stain showed AcLDL could induce apoptosis in THP-1 macrophages in a time and dose dependent manner. Exposure of THP-1 macrophages to 100 microg/ml of AcLDL for 24 hours resulted in a significant increase in caspase 3,7 activity, a significant increase in FC and CE mass of 1.5 and 2.4-fold, meanwhile, a significant increase in transcription factor C/EBP homologous protein and a decrease in Bcl-2 both in protein and mRNA levels were observed with an 8-fold rise of free cholesterol in the ER.</p><p><b>CONCLUSION</b>ER stress is involved in AcLDL induced apoptosis in THP-1 macrophages with free cholesterol accumulation in the ER.</p>