Abstract
Macrophage conversion to atherosclerotic foam cells is partly due to the balance of uptake and efflux of cholesterol. Cholesterol efflux from cells by HDL and its apoproteins for subsequent hepatic elimination is known as reverse cholesterol transport. Numerous methods have been developed to measure in vivo macrophage cholesterol efflux. Most methods do not allow for macrophage recovery for analysis of changes in cellular cholesterol status. We describe a novel method for measuring cellular cholesterol balance using the in vivo entrapment of macrophages in alginate, which retains incorporated cells while being permeable to lipoproteins. Recipient mice were injected subcutaneously with CaCl2 forming a bubble into which a macrophage/alginate suspension was injected, entrapping the macrophages. Cells were recovered after 24 h. Cellular free and esterified cholesterol mass were determined enzymatically and normalized to cellular protein. Both normal and cholesterol loaded macrophages undergo measureable changes in cell cholesterol when injected into WT and apoA-I-, LDL-receptor-, or apoE-deficient mice. Cellular cholesterol balance is dependent on initial cellular cholesterol status, macrophage cholesterol transporter expression, and apolipoprotein deficiency. Alginate entrapment allows for the in vivo measurement of macrophage cholesterol homeostasis and is a novel platform for investigating the role of genetics and therapeutic interventions in atherogenesis.
Highlights
Macrophage conversion to atherosclerotic foam cells is partly due to the balance of uptake and efflux of cholesterol
For the HDL experiments the cells were loaded with 0.1 mg/ml Acetylated human LDL (AcLDL) and 0.3 mM 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (CPTcAMP) for 24 h prior to entrapment in order to stimulate ABCA1 and ABCG1 cholesterol efflux
To confirm that alginate would be suitable for monitoring lipoprotein transport, J774 cells were entrapped in alginate bulbs using alginate concentrations ranging from 0.6% to 1.2%
Summary
Macrophage conversion to atherosclerotic foam cells is partly due to the balance of uptake and efflux of cholesterol. Increased cholesterol efflux is the first step in reverse cholesterol transport (RCT), in which excess cholesteryl ester is hydrolyzed and transported through the plasma membrane by means of ABC transporters to extracellular acceptors, which carry the effluxed cholesterol through the plasma to the liver for conversion to bile acids for subsequent excretion in the feces [3] It is by means of this RCT that HDL and its associated apolipoproteins (e.g., apoA-I and apoE) are thought to exhibit some of their antiatherogenic properties and to furnish the basis of the epidemiologic inverse correlation between HDL levels and coronary artery disease [4,5,6]. The levels of radiolabel in the plasma, liver, bile, and feces are measured over time as a determinant of cholesterol transport out of the macrophages for elimination This protocol has been valuable in determining the effects of apoA-I deletion and overexpression and other participants in HDL metabolism and RCT on cholesterol trafficking from peripheral cells to the feces. The role of other plasma apoproteins and enzymes (e.g., serum amyloid A, cholesteryl ester transfer protein, LCAT), as well as mutant forms of apoA-I (e.g., apoA-I Milano), in RCT have been confirmed in vivo using this method, and as such, it has been the gold standard in confirming in vitro cholesterol efflux results [16,17,18,19]
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