Modification of Low Density Lipoprotein and the Effects of Modified Low Density Lipoproteins on the Endocytic and Secretory Repertoire of the Macrophage

<p>This study examines two aspects of the complex interaction between modified low density lipoprotein (LDL) and macrophages in the initiation and development of the atherosclerotic lesion. The first of these concerns the observed biological modification of LDL that leads to its subsequent enhanced uptake by macrophages, and the role of oxidation in that process. The second aspect of this thesis studies the effects of modified LDL on the functions of macrophages. Studies on the oxidation of LDL showed that LDL isolated from human plasma anticoagulated with EDTA (EDTA-LDL) was 4-fold more resistant to oxidation by reagent H₂O₂, as measured by the thiobarbituric acid (TBA) assay, than LDL prepared from plasma anticoagulated with citrate (CDP-LDL). The LDLs required 1-3 mM H₂O₂ for maximal oxidation by this assay, and ED₅₀s were 1.7 x 10^-3 M for EDTA-LDL and 4.5 x 10^-4M for CDP-LDL. Oxidation was enhanced 2.3-fold by Cu²⁺ ions. Rabbit endothelial cell line monolayers released three orders of magnitude less H₂O₂than was required to oxidize LDL and failed to induce TBA reactivity in either EDTA-LDL or CDP-LDL after a 24-hour coincubation. However, this LDL was subsequently degraded by mouse macrophages more rapidly than untreated LDL. Freshly isolated human monocytes (2 x 10⁶ cells per ml), with or without phorbol myristate acetate (100 ng/ml) to trigger the respiratory burst, did not oxidize LDL in the TBA assay, despite producing large amounts of reactive oxygen intermediates. EDTA-LDL, CDP-LDL, and acetoacetylated LDL failed to trigger H₂O₂ release from human monocytes or macrophages. These results separate oxidation of LDL as measured by TBA assay from the modification of LDL by a rabbit aortic endothelial cell line that leads to its subsequent enhanced degradation by macrophages. Mouse peritoneal macrophages were lipid-loaded by three regimens modeling loading through the scavenger receptor (Ac-LDL cells), by extracellular matrix-bound LDL (DS-LDL cells), and conditions of reduced cholesterol acceptors in the medium (LS/01 cells). Increased cholesterol levels in all three regimens were measured by cholesterol determination, Oil Red O staining of fixed cells, and extraction of Oil Red O from cells and determination of OD₅₃₀. Lipid-laden cells were equal to control macrophages in binding and ingesting immunoglobulin-coated sheep erythrocytes, reflecting Fc-mediated endocytosis. Cellular proteins appeared largely unchanged, but alterations in the secreted protein profile were observed by metabolic labeling with ³⁵S-methionine. DS-LDL loaded cells and LS/01 loaded cells showed increases in a 68 kD protein, and decreases in apo E secretion. Ac-LDL loaded cells showed significant enhancement in apo E secretion. The lipid-laden cells were compared to control cells for three secretory functions of macrophages that could be important in the atherosclerotic plaque. They were still capable of producing all secretory products examined, but the quantities of H₂O₂ and arachidonic acid metabolites are reduced in some cases, and plasminogen activator appeared to be increased. However, we have lipid-loaded the cells for only two days, and foam cells in vivo may be exposed to these conditions for months or years. The different loading regimens result in different changes, which we have separated, but in vivo the cells would be exposed to a complex mixture of native and biologically modified LDL, and LDL complexed glcosaminoglycans and with proteins of the extracellular matrix. It is premature to predict what changes in the macrophage's large repertoire would occur in vivo.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Thesis

Year

1987

Author dc:creator

• Montgomery, Ruth Rebecca

Contributors dc:contributor

• Zanvil Cohn

• modified LDL
• macrophages
• oxidation
• atherosclerosis
• foam cells
• lipid loading
• Life Sciences

Repository record dc:identifier

https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/385

OAI identifier oai:identifier

oai:digitalcommons.rockefeller.edu:student_theses_and_dissertations-1386