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Effect of hyperapo B LDL on cholesterol esterification in THP-1 macrophages
S D Kafonek1, I Raikhel, P S Bachorik
1Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Insights
Hyperapobetalipoproteinemia (hyperapo B) LDL does not promote cellular cholesteryl ester accumulation through LDL receptor-independent pathways. This study found no differences in cellular uptake, degradation, or synthesis between normal and hyperapo B LDL in macrophages.
Area of Science:
- Biochemistry
- Cell Biology
- Cardiovascular Science
Background:
- Hyperapobetalipoproteinemia (hyperapo B) is linked to coronary artery disease and characterized by increased small, dense low-density lipoprotein (LDL) particles.
- The cellular mechanisms driving early atherosclerosis in hyperapo B remain unclear.
Purpose of the Study:
- To investigate whether hyperapo B LDL is preferentially metabolized via LDL receptor-independent pathways.
- To determine if this pathway leads to increased cellular cholesteryl ester (CE) accumulation.
Main Methods:
- THP-1 macrophages, with limited LDL receptor activity, were used to assess non-LDL receptor-mediated lipoprotein uptake.
- LDL from hyperapo B and normal subjects was incubated with THP-1 macrophages to measure CE accumulation.
- LDL particle characteristics (cholesterol-to-protein ratio, density, diameter) were analyzed.
- Cellular uptake, degradation, and CE synthesis rates were measured using radiolabeled assays.
Main Results:
- Hyperapo B LDL exhibited lower cholesterol-to-protein ratio, higher density, and smaller particle diameter compared to normal LDL.
- No significant differences in CE accumulation were observed between normal and hyperapo B LDL in THP-1 macrophages.
- Cellular uptake, degradation, and CE synthesis rates were similar for both normal and hyperapo B LDL in THP-1 and human monocyte-derived (HMD) macrophages.
Conclusions:
- Hyperapo B LDL does not stimulate cellular CE accumulation via LDL receptor-independent pathways in macrophages.
- The metabolism and cellular processing of normal and hyperapo B LDL are comparable in relevant cell models.
Abstract:
Hyperapobetalipoproteinemia (hyperapo B), a common disorder associated with coronary artery disease, is characterized by an increased number of small, dense, low density lipoprotein (LDL) particles. The cellular mechanisms responsible for early atherosclerosis in hyperapo B are unknown. We tested the hypothesis that hyperapo B LDL may be preferentially metabolized through an LDL receptor independent pathway promoting the accumulation of cellular cholesteryl ester (CE). THP-1 macrophages have little inducible LDL receptor activity after differentiation with phorbol esters and are, therefore, suitable for assessing non-LDL receptor mediated uptake of lipoproteins. LDL isolated from hyperapo B donors was found to have significantly lower total cholesterol to protein ratio (P = 0.03), higher average density (P = 0.0001) and smaller particle diameter (P = 0.016) compared with normal (control) LDL. LDL (250 micrograms lipoprotein-protein/ml) from normal (n = 11) and hyperapo B (n = 18) subjects were incubated for 24 h with THP-1 macrophages. The mean (S.D.) CE accumulation was 6.2 (3.6) for the normal and 6.4 (2.6) for the hyperapo B LDL (P = 0.84). CE accumulation in cells incubated with malondialdehyde modified (MDA) LDL, or without added lipoprotein, was 18.2 (2.0) and 0.6 (0.7), respectively. CE mass accumulation was significantly correlated with time (6-48 h) of incubation and concentration (100-500 micrograms/ml) of LDL protein (P < 0.05); no differences were observed between normal and hyperapo B LDL. Similarly, when the major LDL species was isolated by density gradient ultracentrifugation, mean (S.D.) CE was similar for the normal and hyperapo B LDL (8.7 (1.2) vs. 6.9 (1.5)). There were no differences in the mean (S.D.) incorporation of [14C]oleate into CE (nmol/mg cell protein per 6 h) in THP-1 macrophages incubated with normal or hyperapo B LDL (0.238 (0.045) vs. 0.211 (0.046)); results were comparable in human monocyte-derived (HMD) macrophages (0.298 (0.037) vs. 0.258 (0.022)). Also, mean (S.D.) cellular uptake and degradation (ng 125I/mg cell protein per h) in THP macrophages of normal and hyperapo B LDL were similar (uptake: 18 (14) vs. 12 (6.0); degradation: 58 (32) vs. 44 (8)). In summary: (1) hyperapo B LDL did not stimulate the accumulation of cellular CE via LDL receptor independent pathways in THP-1 macrophages, (2) normal and hyperapo B LDL stimulation of CE synthesis is similar in THP-1 and HMD macrophages and (3) no differences in cellular uptake and degradation of normal and hyperapo B LDL were observed in THP macrophages.