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Macrophage uptake of low-density lipoprotein modified by 4-hydroxynonenal. An ultrastructural study
1Department of Vascular Cell Biology and Atherosclerosis Research, Cleveland Clinic Foundation, Ohio.
Abstract:
We have documented the ultrastructural characteristics of the uptake and processing by mouse peritoneal macrophages (MPM) of low-density lipoprotein (LDL) modified with 4-hydroxynonenal (HNE), an intermediate of lipid peroxidation. This was performed as part of a larger biochemical study assessing the role of LDL oxidation in lipid loading of macrophages during atherogenesis. Gold-labeled LDL that was modified with HNE leading to particle aggregation represented the morphologic probe used. When incubated with MPM, the probe became associated with short segments of cell membrane, probably derived from blebs or from lysed cells. At 37 degrees C there was a time-dependent increase in uptake by MPM, and at 4 hours the increase paralleled the degradation by MPM of 125I-labeled HNE-LDL-cAu. Clathrin-coated pits on the cell surface were consistently associated with probe. Uptake of probe appeared to occur via phagocytosis, because pseudopods frequently surrounded probe, and cytochalasin D quantitatively prevented probe uptake. A time-dependent increase was found in the number of gold particles per unit area within vacuoles, some of which were secondary lysosomes, based on acid phosphatase-positive staining. Thus, HNE-induced aggregation of LDL during oxidation, binding of aggregates to clathrin-coated pits on MPM, and subsequent phagocytosis may represent one of the ways lipid-laden foam cells are formed in vivo.
Insights
Modified low-density lipoprotein (LDL) aggregates are taken up by mouse peritoneal macrophages (MPM) via phagocytosis. This process, involving clathrin-coated pits and lysosomes, may contribute to foam cell formation in atherosclerosis.
Area of Science:
- Cell Biology
- Biochemistry
- Pathology
Background:
- Lipid peroxidation generates reactive aldehydes like 4-hydroxynonenal (HNE).
- Oxidized low-density lipoprotein (LDL) plays a critical role in the development of atherosclerosis.
- Macrophages are key immune cells involved in lipid metabolism and foam cell formation.
Purpose of the Study:
- To investigate the ultrastructural mechanisms of modified LDL uptake by macrophages.
- To assess the role of 4-hydroxynonenal (HNE)-modified LDL in macrophage lipid loading.
- To elucidate the cellular processing of HNE-modified LDL by mouse peritoneal macrophages (MPM).
Main Methods:
- Utilized gold-labeled HNE-modified LDL as a morphologic probe.
- Incubated the probe with MPM at 37 degrees C and observed time-dependent uptake.
- Employed electron microscopy, biochemical assays (125I-LDL degradation), and cytochalasin D treatment to study uptake mechanisms.
- Identified cellular compartments involved using acid phosphatase staining for secondary lysosomes.
Main Results:
- HNE-modified LDL aggregated and associated with MPM cell membranes, likely from blebs or lysed cells.
- Time-dependent uptake of HNE-LDL by MPM paralleled its degradation.
- Uptake occurred via phagocytosis, evidenced by pseudopod engulfment and cytochalasin D inhibition.
- Gold particles were found within vacuoles, including secondary lysosomes, in a time-dependent manner.
- Clathrin-coated pits were consistently involved in probe association with the cell surface.
Conclusions:
- HNE-induced LDL aggregation facilitates binding to macrophage clathrin-coated pits.
- Phagocytosis of aggregated HNE-LDL by MPM is a significant uptake mechanism.
- This pathway represents a potential mechanism for foam cell formation in vivo during atherogenesis.
