Alginic Acid Cell Entrapment: A Novel Method for Measuring In Vivo Macrophage Cholesterol Homeostasis

Cardiovascular disease including atherosclerosis continues to be one of the leading causes of mortality throughout the world (1). The accumulation of cholesterol-loaded macrophages is a critical step in the progression of atherosclerotic lesion development. This accumulation involves the uptake of free and esterified cholesterol by the cells from modified LDL (2). The excess free cholesterol is converted to cholesteryl ester by the enzymatic activity of ACAT and stored in lipid droplets. As cholesterol loading plays a critical role in atherosclerosis progression, there is great interest in understanding factors that prevent cholesterol accumulation either through decreased cellular cholesterol uptake or increased cholesterol efflux. Cholesterol loading engenders a complex network of changes in gene and protein expression, mostly furthering inflammation. 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 (4–6). There is an extensive body of literature demonstrating the in vitro impact of apoA-I, HDL, and cholesterol transporters on macrophage cholesterol levels (7, 8). Until recently, however, there have been no in vivo methods to examine the impact of RCT on the macrophages themselves, in other words, on the cholesterol balance within the macrophage foam cells. Recent work by Weibel and colleagues (9) has demonstrated the importance of considering both cholesterol influx and efflux when evaluating the impact of serum components on cholesterol homeostasis and atherosclerosis. The most widely used method for measuring in vivo macrophage cholesterol efflux has been the radiolabel method developed by the Rader group (10, 11). In this method, macrophages are loaded ex vivo with radiolabeled forms of nonesterified cholesterol and subsequently injected into the peritoneal cavity of the recipient animals. Variations of the protocol are also used in which the cells are injected subcutaneously rather than into the peritoneal cavity (12, 13). The levels of radiolabel in the plasma, liver, bile, and feces are then 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. This protocol was originally developed using J774 cultured mouse macrophages; however, it is also suitable for use with primary macrophages overexpressing or lacking lipid transporters. The protocol has been used to show that ABC cholesterol transporters ABCA1 and ABCG1 promote in vivo macrophage cholesterol efflux, confirming not only the function of these transport proteins in culture but also the additive impact of their activity (14, 15). The scavenger receptor class B type I (SR-BI), however, was shown to have no in vivo macrophage cholesterol efflux activity using this method, despite in vitro results indicating it does promote cholesterol efflux (15). 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 (16–19). Other methods for determining in vivo macrophage cholesterol efflux have been examined including the centripetal cholesterol flux method in which dual radioisotopes of cholesterol precursors and labeled LDL cholesterol are administered and used to estimate the cholesterol efflux from the peripheral cells, with the assumption that the synthesis of cholesterol and uptake by peripheral cells is equal to the efflux (20). However, this method failed to show an effect of apoA-I and HDL on in vivo cholesterol transport and may not be specific enough to reveal cholesterol transport effects unique to macrophages (21–23). Recently, Turner et al. (24) have used constant infusion of [13C]cholesterol in humans to monitor the tissue free cholesterol efflux, esterification of free cholesterol in the plasma, and excretion of plasma-derived free cholesterol as fecal sterols. The measurement of fecal steroid levels has also been used to compare RCT on different genetic backgrounds or following interventions aimed at increasing RCT. The administration of apoA-I increased fecal steroid content in human studies, while the deletion of ABCA1 in mice had no effect on fecal sterol output (22, 25). Despite the usefulness of the radiolabeled macrophage injection method, there are drawbacks. Recent studies suggest that the radiolabeled cholesterol does not accurately reflect the total cholesterol pool found in the macrophages (26). As only the injected macrophages are radiolabeled, a combination of radiolabel efflux and changes in cellular cholesterol mass are necessary to measure the net ingress/egress of cholesterol from the cells over the course of the experiment (9). Finally, as the injected cells move out of the peritoneal cavity, they are not recoverable for measurement of cellular cholesterol homeostasis. Weibel and colleagues (27) have addressed these issues with a novel method for assessing macrophage cholesterol trafficking in which nonlabeled macrophages are inserted into hollow fibers (0.2 µm pore size), which are then surgically implanted into the mouse peritoneal cavity for recovery after 24-48 h. They demonstrated that this method allows for modification of both macrophage and recipient genetic background, recovery of macrophages, and measurement of free and esterified cholesterol mass from the recovered macrophages without the need of radiolabel. Given the pore size of the fibers used, LDL particles were able to pass through the fiber allowing for evaluation of net uptake/efflux of cholesterol. We attempted to use this method and found the injection of the recommended 100 µl (3.5 × 106 cells) to be very difficult in 2 cm of 1 mm inner diameter Microkros hollow fiber, as was the recovery of viable cells. Given that the fiber is cylindrical, the total inner volume of the fiber [π × 20 mm × (0.5 mm)2] would be 15.7 mm3 or 15.7 µl. Additionally, this method requires surgical insertion of the hollow fibers rather than the simple injection of macrophages used with the Rader method. We here present a novel method for assessing macrophage participation in cholesterol homeostasis that satisfies the above concerns. The method involves the in vivo entrapment of the macrophages in alginate. Alginates are naturally occurring polysaccharides derived from seaweed made up of copolymers of nonbranched β-d-mannuronic acid and α-l-guluronic acid (28). The addition of a divalent cation such as calcium to alginic acid solution results in the formation of a gel of increasing rigidity depending on alginate concentration. The gel can additionally be disrupted with the addition of a citrate buffer to chelate the calcium ions. Because of its high biocompatibility, alginate is the most common material used in entrapment of islet cells for transplantation, with alginate implants being at least 50% recoverable after as long as 12 weeks in vivo (29). This combination of biocompatibility, stability, and ease of cell entrapment and harvest suggested that entrapment of macrophages in alginate might be a suitable method for the in vivo measurement of macrophage cholesterol homeostasis with the subsequent recovery and analysis of macrophages. This alginate-based method that we describe requires no surgery, and the macrophages are readily recovered at >95% cell viability.