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Mechanisms of enhanced macrophage apoE secretion by oxidized LDL
A A Cader1, F M Steinberg, T Mazzone
1Department of Medicine, University of Washington, Seattle 98195-6426, USA.
Abstract:
Previous studies have demonstrated that atherosclerotic lesions contain apoE synthesized primarily by macrophages. As oxidized LDL has been implicated in the development of atherosclerosis, its effect on macrophage apoE synthesis and secretion was examined. Human monocytic leukemia cells, THP-1, and human monocyte-derived macrophages were exposed to various forms of oxidatively modified LDL for determination of their effect on apoE mRNA and protein levels. Extensively copper oxidized (Cu-oxidized) LDL resulted in a time- and concentration-dependent increase in apoE mRNA and protein as compared to other forms of oxidized LDL, i.e., LDL modified by soybean lipoxygenase (SLO), azoamidinopropane HCl (AAPH), and hypochlorite (HOCl). Consistent with these results, experiments using THP-1 cells transfected with the apoE promoter linked to a luciferase reporter gene indicated that Cu-oxidized LDL was the most potent stimulator of apoE transgene expression. Enhanced apoE expression due to Cu-oxidized LDL was shown to be due to cholesterol accumulation as well as additional factors. HPLC analysis of the various forms of modified LDL revealed that 7-ketocholesterol was the major oxysterol present in Cu-oxidized LDL. AAPH-oxidized LDL contained significantly less 7-ketocholesterol than Cu-oxidized LDL and virtually no 7-ketocholesterol was detected in SLO- or HOCl-oxidized LDL. Northern blot analysis indicated an increase in apoE mRNA in response to increasing concentrations of 7-ketocholesterol. These results elucidate a potential role of oxidized LDL, and specifically 7-ketocholesterol, in the stimulation of macrophage apoE secretion in atherosclerotic lesions.
Insights
Copper-oxidized LDL significantly increases macrophage apolipoprotein E (apoE) production, primarily due to 7-ketocholesterol. This finding highlights a key mechanism in atherosclerosis development and apoE secretion.
Area of Science:
- Biochemistry
- Immunology
- Cardiovascular Research
Background:
- Atherosclerotic lesions characteristically contain apolipoprotein E (apoE), mainly synthesized by macrophages.
- Oxidized low-density lipoprotein (LDL) is a known factor in atherosclerosis development.
Purpose of the Study:
- To investigate the impact of various oxidized LDL forms on macrophage apoE synthesis and secretion.
- To identify specific components within oxidized LDL responsible for modulating apoE expression.
Main Methods:
- Exposure of human leukemia cells (THP-1) and monocyte-derived macrophages to different forms of oxidized LDL (Cu-oxidized, SLO-oxidized, AAPH-oxidized, HOCl-oxidized).
- Quantification of apoE mRNA and protein levels.
- Luciferase reporter gene assay to assess apoE promoter activity.
- High-Performance Liquid Chromatography (HPLC) to analyze oxysterol content, specifically 7-ketocholesterol.
- Northern blot analysis to correlate 7-ketocholesterol levels with apoE mRNA expression.
Main Results:
- Copper-oxidized LDL (Cu-oxidized LDL) demonstrated a significant, time- and concentration-dependent increase in both apoE mRNA and protein levels compared to other oxidized LDL variants.
- Cu-oxidized LDL was the most potent stimulator of apoE transgene expression, as indicated by luciferase reporter assays.
- Cholesterol accumulation and other factors contributed to enhanced apoE expression induced by Cu-oxidized LDL.
- 7-ketocholesterol was identified as the predominant oxysterol in Cu-oxidized LDL, with significantly lower levels in other oxidized LDL forms.
- Northern blot analysis confirmed a direct correlation between increasing 7-ketocholesterol concentrations and elevated apoE mRNA levels.
Conclusions:
- Oxidized LDL, particularly Cu-oxidized LDL, plays a crucial role in stimulating macrophage apoE secretion.
- 7-ketocholesterol is a key mediator in the process, driving the upregulation of apoE expression in macrophages within atherosclerotic lesions.