Endoplasmic reticulum stress is involved in acetylated low-density lipoprotein induced apoptosis in THP-1 differentiated macrophages.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
ER stress is involved in AcLDL induced apoptosis in THP-1 macrophages with free cholesterol accumulation in the ER.
Division of Nephrology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China.
Jian-ling
JL
Tao
Xiong-zhong
XZ
Ruan
Hang
H
Li
Xue-mei
XM
Li
John F
JF
Moorhead
Zac
Z
Varghese
Xue-wang
XW
Li
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects*; D018613:Microscopy, Confocal; D016133:Polymerase Chain Reaction; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D051742:Transcription Factor CHOP / Q000235:genetics
C497249:DDIT3 protein, human; D008077:Lipoproteins, LDL; D019253:Proto-Oncogene Proteins c-bcl-2; C032629:acetyl-LDL; D051742:Transcription Factor CHOP; D002784:Cholesterol