Ginny Kellner-Weibel

Importance of Different Pathways of Cellular Cholesterol Efflux

The removal of excess free cholesterol from cells by HDL or its apolipoproteins is important for maintaining cellular cholesterol homeostasis. This process is most likely compromised in the atherosclerotic lesion because the development of atherosclerosis is associated with low HDL cholesterol. Multiple mechanisms for efflux of cell cholesterol exist. Efflux of free cholesterol via aqueous diffusion occurs with all cell types but is inefficient. Efflux of cholesterol is accelerated when scavenger receptor class-B type I (SR-BI) is present in the cell plasma membrane. Both diffusion-mediated and SR-BI-mediated efflux occur to phospholipid-containing acceptors (ie, HDL and lipidated apolipoproteins); in both cases, the flux of cholesterol is bidirectional, with the direction of net flux depending on the cholesterol gradient. The ATP-binding cassette transporter AI (ABCA1) mediates efflux of both cellular cholesterol and phospholipid. In contrast to SR-BI-mediated flux, efflux via ABCA1 is unidirectional, occurring to lipid-poor apolipoproteins. The relative importance of the SR-BI and ABCA1 efflux pathways in preventing the development of atherosclerotic plaque is not known but will depend on the expression levels of the two proteins and on the type of cholesterol acceptors available.

A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol

Studies have shown a negative association between cellular cholesterol efflux and coronary artery disease (CAD). Standard protocol for quantitating cholesterol efflux involves labeling cells with [3H]cholesterol and measuring release of the labeled sterol. Using [3H]cholesterol is not ideal for the development of a high-throughput assay to screen large numbers of serum as would be required in studying the link between efflux and CAD. We compared efflux using a fluorescent sterol (boron dipyrromethene difluoride linked to sterol carbon-24, BODIPY-cholesterol) with that of [3H]cholesterol in J774 macrophages. Fractional efflux of BODIPY-cholesterol was significantly higher than that of [3H]cholesterol when apo A-I, HDL3, or 2% apoB-depleted human serum were used as acceptors. BODIPY-cholesterol efflux correlated significantly with [3H]cholesterol efflux (p < 0.0001) when apoB-depleted sera were used. The BODIPY-cholesterol efflux correlated significantly with preβ-1 (r2 = 0.6) but not with total HDL-cholesterol. Reproducibility of the BODIPY-cholesterol efflux assay was excellent between weeks (r2 = 0.98, inter-assay CV = 3.31%). These studies demonstrate that BODIPY-cholesterol provides an efficient measurement of efflux compared with [3H]cholesterol and is a sensitive probe for ABCA1-mediated efflux. The increased sensitivity of BODIPY-cholesterol assay coupled with the simplicity of measuring fluorescence results in a sensitive, high-throughput assay that can screen large numbers of sera, and thus establish the relationship between cholesterol efflux and atherosclerosis.

SERUM ALBUMIN ACTS AS A SHUTTLE TO ENHANCE CHOLESTEROL EFFLUX FROM CELLS

An important mechanism contributing to cell cholesterol efflux is aqueous transfer in which cholesterol diffuses from cells into the aqueous phase and becomes incorporated into an acceptor particle. Some compounds can enhance diffusion by acting as shuttles transferring cholesterol to cholesterol acceptors, which act as cholesterol sinks. We have examined whether particles in serum can enhance cholesterol efflux by acting as shuttles. This task was accomplished by incubating radiolabeled J774 cells with increasing concentrations of lipoprotein-depleted sera (LPDS) or components present in serum as shuttles and a constant amount of LDL, small unilamellar vesicles, or red blood cells (RBC) as sinks. Synergistic efflux was measured as the difference in fractional efflux in excess of that predicted by the addition of the individual efflux values of sink and shuttle alone. Synergistic efflux was obtained when LPDS was incubated with cells and LDL. When different components of LPDS were used as shuttles, albumin produced synergistic efflux, while apoA-I did not. A synergistic effect was also obtained when RBC was used as the sink and albumin as shuttle. The previously observed negative association of albumin with coronary artery disease might be linked to reduced cholesterol shuttling that would occur when serum albumin levels are low.

Update on HDL Receptors and Cellular Cholesterol Transport

Efflux is central to maintenance of tissue and whole body cholesterol homeostasis. The discovery of cell surface receptors that bind high-density lipoprotein (HDL) with high specificity and affinity to promote cholesterol release has significantly advanced our understanding of cholesterol efflux. We now know that 1) cells have several mechanisms to promote cholesterol release, including a passive mechanism that depends on the physico-chemical properties of cholesterol molecules and their interactions with phospholipids; 2) a variety of HDL particles can interact with receptors to promote cholesterol transport from tissues to the liver for excretion; and 3) interactions between HDL and receptors show functional synergy. Therefore, efflux efficiency depends both on the arrays of receptors on tissue cells and HDL particles in serum.

In Vitro Studies and Mass Flux of Cholesterol Between Serum and Macrophages

Reverse cholesterol transport (RCT) is a complex process. Several steps, utilizing enzymes and transfer proteins, occur within the plasma compartment before the cholesterol molecules are delivered from peripheral cells to hepatocytes (Atherosclerosis 88:99-107, 1991; Arterioscler Thromb Vasc Biol 21:13-27, 2001). Both intracellular and extracellular factors affect the direction of cholesterol flux between cells and serum. When cells are incubated with serum in vitro there is flux of free cholesterol (FC) out of the cell (efflux) while simultaneously there is movement of lipoprotein FC and cholesteryl ester (CE) into the cell (influx). Net cholesterol flux is the difference between influx and efflux and this difference may result in net accumulation, net depletion, or no change in cell cholesterol content. The protocols to measure the efflux of labeled cholesterol are relatively simple since only FC undergoes efflux and the assay can be easily adapted for high throughput use. A significant relationship between net cholesterol removal and the fractional efflux of tracer cholesterol is seen in cholesterol-enriched cells indicates that in vitro isotopic measures of serum efflux efficiency may be valid surrogate measure of the capacity of human serum to remove excess tissue cholesterol in vivo. We have also measured net cholesterol flux as the change in cell cholesterol content upon incubation of J774 cells and mouse peritoneal macrophages with human serum. We found that the cholesterol content of the cell modulates the relative contribution of efflux pathways (ABCAI, SRBI, ABCGI) and thus changes the cell’s response to HDL subfractions and the direction of net cholesterol flux.