Abstract
The uptake of oxidized low density lipoprotein (oxLDL) by macrophages leads to foam cell formation and fatty streaks, which represent early sites of potential atheroma development. We developed a cell culture model of chronic oxLDL exposure to determine whether hallmark parameters of oxLDL uptake and cytotoxicity are altered during foam cell formation and to determine changes in protein and mRNA expression that distinguish acute and chronic oxLDL exposure. Although the extent of oxLDL uptake did not change, a resistance to oxLDL-induced cytotoxicity was observed in the chronically exposed cells. Macrophages that have been chronically exposed to oxLDL required a 40% higher concentration of oxLDL to achieve 50% survival in a 48-h treatment relative to macrophages subjected to a single oxLDL exposure. A main feature of the differentially expressed proteome was a series of significantly overexpressed antioxidant and antioxidant-related proteins in the oxLDL-exposed cells. A large proportion of these proteins (45%) was overexpressed in the chronically exposed cells prior to the oxLDL treatment, indicative of the unique phenotype produced by the chronic treatment. Analysis of the transcriptome also revealed a broad increase in the expression of antioxidant and antioxidant-related proteins. In addition, the transcriptome experiments found an increased inflammatory response under conditions of both acute and chronic oxLDL exposure. Overall the combined functional, proteomic, and transcriptomic experiments show that macrophages respond to oxLDL by developing an oxidative stress resistance that increases and stabilizes with chronic exposure. Furthermore this protective response and the increased foam cell survival that it supports amplifies their proatherogenic role by promoting a continued inflammatory state.
Highlights
The uptake of oxidized low density lipoprotein by macrophages leads to foam cell formation and fatty streaks, which represent early sites of potential atheroma development
Development of J774-CE Macrophages—The effects of chronic oxidized low density lipoprotein (oxLDL) exposure on J774 macrophages were studied by exposing cells to 50 g/ml oxLDL for h twice per week for 3 months, the equivalent of oxLDL exposures
No differences were observed in the morphology, growth rate, or cell cycle progression of the J774-CE cells relative to the parent J774 cells. The intent of these experiments was to investigate the effects of chronic oxLDL exposure on the two hallmarks of the oxidative hypothesis of atherosclerosis: oxLDL internalization and cytotoxicity
Summary
Many factors contribute to the progression of atherosclerosis, foam cells appear to have a uniquely proatherogenic phenotype that goes beyond the lipid deposition that is produced by the unregulated LDL uptake. A proteomic approach has not been applied, to the cellular proteins of foam cells Those previous experiments used only a single oxLDL exposure, so the effect of chronic oxLDL exposure on the foam cell phenotype is unknown. Other models of chronic exposure to agents such as ethanol [21] and hydrogen peroxide [22, 23] have identified important differences in protein expression, relative to the acute exposure conditions, that contributed to distinct cellular phenotypes. A system biology approach has been used to characterize this chronic exposure model and test the hypothesis that changes in foam cell phenotype are produced by long term exposure to oxLDL relative to both untreated macrophages and foam cells formed by a single oxLDL exposure
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