Abstract
BackgroundThe transformation of normal macrophage cells into lipid-laden foam cells is an important step in the progression of atherosclerosis. One major contributor to foam cell formation in vivo is the intracellular accumulation of cholesterol.MethodsHere, we report the effects of various combinations of low-density lipoprotein, sterols, lipids and other factors on human macrophages, using an automated image analysis program to quantitatively compare single cell properties, such as cell size and lipid content, in different conditions.ResultsWe observed that the addition of cholesterol caused an increase in average cell lipid content across a range of conditions. All of the sterol-lipid mixtures examined were capable of inducing increases in average cell lipid content, with variations in the distribution of the response, in cytotoxicity and in how the sterol-lipid combination interacted with other activating factors. For example, cholesterol and lipopolysaccharide acted synergistically to increase cell lipid content while also increasing cell survival compared with the addition of lipopolysaccharide alone. Additionally, ergosterol and cholesteryl hemisuccinate caused similar increases in lipid content but also exhibited considerably greater cytotoxicity than cholesterol.ConclusionsThe use of automated image analysis enables us to assess not only changes in average cell size and content, but also to rapidly and automatically compare population distributions based on simple fluorescence images. Our observations add to increasing understanding of the complex and multifactorial nature of foam-cell formation and provide a novel approach to assessing the heterogeneity of macrophage response to a variety of factors.
Highlights
The transformation of normal macrophage cells into lipid-laden foam cells is an important step in the progression of atherosclerosis
We are interested in investigating the parameters contributing to the second of these steps, during which macrophages are transformed into foam cells, and in applying a novel computational method to assess the heterogeneity of the cellular response to a variety of factors
In order to be eliminated from the cell, the accumulated free cholesterol must be reesterified by enzymes such as sterol O-acyltransferase (SOAT, known as acyl-CoA cholesterol acyltransferase – ACAT) in a process regulated by the liver X receptor (LXR) and the retinoid X receptor (RXR) [7, 8]
Summary
The transformation of normal macrophage cells into lipid-laden foam cells is an important step in the progression of atherosclerosis. The uptake of cholesterol (predominantly in the form of cholesterol esters encapsulated in LDL) is accelerated by membrane proteins, including scavenger receptors scavenger receptor A (SRA), CD36 and CD68, resulting in the internalization of cholesterol esters that are broken down to free cholesterol in lysosomes [4, 5]. As this exogenous cholesterol accumulates within the cell, the endogenous cholesterol synthesis pathway – through the sterol regulatory elementbinding proteins (SREBPs) – is suppressed [6]. If exogenous cholesterol accumulates too quickly within a cell, it can overwhelm the LXR-regulated reverse transport pathway and result in the buildup of large quantities of cholesterol and associated lipids – potentially resulting in excessive lipid droplet formation, upregulation of a number of inflammatory factors and cell death [9]
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