FTY720 Stimulates 27-Hydroxycholesterol Production and Confers Atheroprotective Effects in Human Primary Macrophages
Rationale: The synthetic sphingosine analog FTY720 is undergoing clinical trials as an immunomodulatory compound, acting primarily via sphingosine 1-phosphate receptor activation. Sphingolipid and cholesterol homeostasis are closely connected but whether FTY720 affects atherogenesis in humans is not known.
Objective: We examined the effects of FTY720 on the processing of scavenged lipoprotein cholesterol in human primary monocyte-derived macrophages.
Methods and Results: FTY720 did not affect cholesterol uptake but inhibited its delivery to the endoplasmic reticulum, reducing cellular free cholesterol cytotoxicity. This was accompanied by increased levels of Niemann–Pick C1 protein (NPC1) and ATP-binding cassette transporter (ABC)A1 proteins and increased efflux of endosomal cholesterol to apolipoprotein A-I. These effects were not dependent on sphingosine 1-phosphate receptor activation. Instead, FTY720 stimulated the production of 27-hydroxycholesterol, an endogenous ligand of the liver X receptor, leading to liver X receptor–induced upregulation of ABCA1. Fluorescently labeled FTY720 was targeted to late endosomes, and the FTY720-induced upregulation of ABCA1 was NPC1-dependent, but the endosomal exit of FTY720 itself was not.
Conclusions: We conclude that FTY720 decreases cholesterol toxicity in primary human macrophages by reducing the delivery of scavenged lipoprotein cholesterol to the endoplasmic reticulum and facilitating its release to physiological extracellular acceptors. Furthermore, FTY720 stimulates 27-hydroxycholesterol production, providing an explanation for the atheroprotective effects and identifying a novel mechanism by which FTY720 modulates signaling.
The synthetic sphingosine analog 2-amino-2-[2-(4-n-octylphenyl)ethyl]-1,3-propanediol hydrochloride (FTY720) is undergoing clinical trials for the treatment of immunologic disorders.1 It is a prodrug phosphorylated by type 2 sphingosine kinase to form FTY720-phosphate (FTY720-P).2 FTY720-P is an analog of sphingosine 1-phosphate (S1P) that mediates its immunomodulatory effects primarily through G protein–coupled S1P receptors.3,4 The S1P1 receptor regulates the egress of lymphocytes from secondary lymphoid organs. By inducing the internalization and degradation of the S1P1 receptor, FTY720-P reduces circulating lymphocyte counts, and is therefore considered a promising therapy for immune disorders, particularly multiple sclerosis.5 Sphingosine serves not only as a bioactive signaling lipid, but also constitutes the backbone of all sphingolipids that are structural components of eukaryotic cell membranes. Sphingolipids are thought to associate with cholesterol to form membrane rafts that are involved in multiple cellular functions, including membrane trafficking and signaling.6,7
Atherosclerotic lesions are characterized by the accumulation of cholesterol in the arterial intima. The lesions form when modified low-density lipoproteins (LDLs) are trapped in the intima and elicit an inflammatory response, attracting inflammatory cell types, in particular monocyte-derived macrophages, to scavenge the particles. Because this leads to excessive cholesterol uptake, macrophages are transformed into lipid-laden foam cells.8 Such cells are found already in early atheromas, called fatty streaks. In advanced lesions, foam cells become overloaded with free cholesterol and undergo cell death, forming the necrotic core of the lesion that becomes prone to rupture.9
On a Western diet, fatty streaks are present already from childhood, and thus probably also in individuals receiving FTY720 therapy. Considering the long-term need of immunomodulatory therapies in many diseases, it is important to assess if FTY720 affects atherogenesis. To our knowledge, this has not been investigated in humans. The findings are in part contradictory in animals. Two studies reported a protective effect of FTY720 in murine atherosclerosis, using LDL-receptor or apolipoprotein (apo)E-deficient mice on a high-fat diet.10,11 The effect was suggested to result from decreased recruitment of inflammatory cells into the lesions because of reduced S1P receptor activation. However, in apoE−/− mice on a normal diet, no changes in atherosclerotic lesions were observed but the animals developed hypercholesterolemia.12
Here, we investigated whether FTY720 affects cholesterol processing in human primary monocyte-derived macrophages deriving from healthy individuals. The results provide evidence that FTY720 protects macrophages from cholesterol toxicity and identify a new mechanism by which this compound affects lipid signaling.
Macrophage serum-free medium was from Gibco and recombinant human granulocyte–macrophage colony stimulating factor from Invitrogen. Other cell culture media were from Sigma-Aldrich. The anti-NPC-1 antibody was from Novus Biologicals; anti–lysosome-associated membrane protein (LAMP)-1 (H4A3) was from Developmental Studies Hybridoma Bank; anti–ATP-binding cassette transporter (ABC)A1 was from Abcam; anti–C/EBP-homologous protein (CHOP) (B-3) was from Santa Cruz Biotechnology; and secondary antibodies were from Invitrogen. LDL was provided by Dr Matti Jauhiainen (National Public Health Institute, Helsinki, Finland), and lipid-free apoA-I was provided by Dr Peter Lerch (Swiss Red Cross, Bern, Switzerland). DiI-acLDL was from Invitrogen, [3H]oleic acid, [3H]cholesterol oleate, [14C]cholesterol oleate and [14C]oleoyl coenzyme A from Amersham. BODIPY-FTY720 was synthesized as described.13
Cell Culture and Viability
Human monocytes from healthy control subjects (in total, from 43 individuals) were isolated from buffy coat cells (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland) as described.14 Monocytes were allowed to adhere to the flask for 1 hour in RPMI medium, washed once with PBS and differentiated into macrophages for one week in macrophage serum-free medium plus 10 ng/mL granulocyte–macrophage colony stimulating factor plus 100 U/mL penicillin and 100 μg/mL streptomycin. CHO cells were cultured in a 1:1 solution of Ham’s F12 and D-MEM supplemented with 5% FBS plus 100 U/mL penicillin and 100 μg/mL streptomycin. FTY720 was added to cells from DMSO and control samples received DMSO only. DMSO concentration did not exceed 0.1%. Cell viability was measured using the CellTiter 96 Aqueous assay (Promega).
Cells were washed three times with PBS and harvested in 1% NP-40 in PBS plus protease inhibitors (chymostatin, leupeptin, antipain, and pepstatin A at 25 μg/mL). Protein concentrations were determined using the Bio-Rad protein assay kit. Cholesterol and cholesteryl ester concentrations were determined using the Amplex red cholesterol assay kit (Invitrogen) from amounts corresponding to 10 μg of protein. Radiolabeling was carried out by incubating acLDL (1.5 mg/mL) with 1:10 volume of [3H]cholesterol in DMSO (40 μCi [3H]cholesterol/mg acLDL). The solution was incubated for 2 hours at 40°C and dialyzed against 150 mmol/L NaCl plus 1 mmol/L EDTA, pH 7.4. Oxysterol determinations were performed as described.15
Cells were labeled with [3H]oleic acid in serum-free medium containing 2% fatty acid–free BSA for 6 hours, washed 3 times with PBS and scraped into a Kimax tube in 2% NaCl. Samples of cell suspensions were taken for protein determinations and [3H]oleate incorporation into cholesteryl esters was measured as described.16
Acyl-Coenzyme A Cholesterol Acyltransferase Activity
Cells were washed with ice-cold PBS and scraped, and the pellet was resuspended in Buffer A (20 mmol/L Tris-HCl, 1 mmol/L EDTA; pH 7.7). The suspension was passed through a 25 gauge needle 10 times for homogenization. Aliquots of the homogenate were used for protein determination. A 40 μL aliquot was mixed with 10 μL Buffer A plus 2 mg/mL fatty acid–free BSA plus 250 μg/mL cholesterol. The solution was incubated at 37°C for 2 minutes and the reaction started by adding 50 μL of Buffer A plus 2 mg/mL fatty acid–free BSA plus [14C]oleoyl coenzyme A. The solution was vortexed briefly, incubated for 10 minutes at 37°C and the reaction stopped by adding methanol (1 mL) and CHCl3 (1 mL). [3H]Cholesteryl oleate was added as an internal standard and cholesteryl esters measured as described.16
Cells were in CO2-independent medium at 37°C during microscopy. For wide-field microscopy, an Olympus IX71 microscope with a UAPO×40/NA 1.35 oil objective was used. TILL Photonics imago QE camera and TILL Vision v4.01 software were used for image acquisition. Confocal microscopy was performed using a Leica DM RXA2/TCS SP2 microscope with a HCX APO 63x/NA 0.9 water objective. Leica Confocal Scanning software was used for image acquisition.
Macrophages on coverslips were fixed, osmificated, and embedded, and the cells were sectioned horizontally. Multilamellar bodies/cell profile were counted. For quantification of mitochondrial–late endosomal compartment (LE) contacts, every fourth cell was photographed and the number of mitochondria making contact with LEs versus the total number of mitochondria/cell profile were counted. Late endosomes/lysosomes were identified by morphological criteria as described.17 Contact was defined as a proximity of ≤50 nm between membranes.
Proteins were separated by SDS-PAGE (15 μg/lane; 6% to 15% gels depending on protein size), transferred to nitrocellulose membrane and incubated with primary antibodies at 4°C overnight and secondary antibodies for 45 minutes at room temperature. After extensive washing with 0.1% Tween in Tris-buffered saline, the blots were developed using the Enhanced Chemiluminiscense kit (Amersham).
Results are expressed as means±SEM from a minimum of 3 independent experiments. Statistical analysis was made using Student’s t test for paired observations. When 3 or more means were tested, 1-way ANOVA was performed, followed by Dunnett’s test for multiple comparisons against a single control. Statistical significance (P<0.05) is denoted with an asterisk.
FTY720 Affects Cholesterol Deposition in Human Monocyte–Derived Macrophages and Protects Them From Death Induced by Cholesterol Overload
Foam cell formation in human macrophages was induced by incubating them with 50 μg/mL acetylated LDL (acLDL) for 1 to 3 days. This induces a several-fold increase in cellular cholesteryl esters, whereas free (unesterified) cholesterol increases only moderately (Figure 1A). Macrophages treated with 1 μmol/L FTY720 displayed a significant reduction in the amount of cholesteryl esters and a tendency toward increased free cholesterol on acLDL loading as compared to control (vehicle only) cells (Figure 1A). At 3 days of acLDL loading, there was less total cholesterol in FTY720-treated cells (30.2±2.7 ng/μg protein) than in control cells (33.0±2.3 ng/μg protein) (n=12 individuals, P<0.05).
These data indicate that FTY720 does not affect the extent of cholesterol uptake from modified LDL but suggest a stimulatory effect on cholesterol removal and an inhibitory effect on cholesterol esterification. In support of the latter, we found that FTY720 led to a reduction in the amount of [3H]oleic acid incorporated into cholesteryl esters in acLDL-loaded macrophages (Figure 1B). Importantly, the activity of acyl-coenzyme A cholesteryl acyl transferase (ACAT) in macrophage lysates was not affected (Figure 1C), suggesting that FTY720 impairs the delivery of cholesterol to ACAT in the endoplasmic reticulum (ER), but not the enzyme activity per se.
FTY720 may induce cell death at high concentrations.18 We found that 1 μmol/L FTY720 did not impair macrophage viability (Figure 1D). To the contrary, when macrophages were challenged with free cholesterol overloading as in,19 FTY720-treated cells were more resistant to death (Figure 1D and 1E) and showed decreased induction of the unfolded protein response mediator CHOP (Figure 1F). Together, these results indicate that FTY720 protects macrophages from free cholesterol-induced toxicity and suggest that this protection results from inhibition of cholesterol transport to the ER.
FTY720 Reduces the Incorporation of acLDL-Derived Cholesterol into Cholesteryl Esters and Stimulates its Release to Apolipoprotein A-I
To specifically address how FTY720 affects cholesterol that enters macrophages via acLDL, we incubated the cells for 4 hours with [3H]cholesterol-labeled acLDL, followed by a 3-hour chase in the presence of the extracellular cholesterol acceptor apoA-I. FTY720-treated cells took up similar amounts of [3H]cholesterol as control cells (Figure 2A), in agreement with the results with unlabeled cholesterol. However, [3H]cholesterol was more avidly released from FTY720-treated cells to apoA-I (Figure 2A and 2C). Moreover, the esterification of [3H]cholesterol was reduced in FTY720 treated cells (Figure 2B), in accordance with the earlier results (Figure 1).
To study the effect of FTY720 on the removal of the foam cell cholesterol mass to apoA-I, we incubated macrophages with acLDL for 3 days as in Figure 1, followed by a 3-day incubation with apoA-I. We found that after incubation with apoA-I, control and FTY720-treated cells reached similar levels of both free and esterified cholesterol (Figure 2D). Taken together, these data show that FTY720 induced a shift in the handling of acLDL derived cholesterol from esterification toward efflux from cells.
Effects of FTY720 on Late Endosomes and on Proteins Involved in Cholesterol Exit From Late Endosomes
We found that FTY720 induced a significant increase in the amounts of Niemann–Pick C1 protein (NPC1) and ATP-binding cassette transporter A1 (ABCA1) (Figure 3), 2 large multitransmembrane proteins involved in the exit of cholesterol from late endosomal compartments (LEs) and in its efflux to apoA-I, respectively.20,21 Notably, the levels of the soluble sterol-binding Niemann–Pick C2 protein (NPC2) were not affected by FTY720 (Figure 3), although NPC1 and NPC2 are considered to function in the same pathway.22
To further address the endosomal effects of FTY720, we investigated the ultrastructure of the LEs. We found that FTY720 induced a proliferation of LEs, as judged by the increased immunoreactivity against LAMP-1 in acLDL-loaded cells (Figure 4A) and an increased number of multilamellar bodies as assessed by electron microscopy (Figure 4B). The LEs of FTY720-treated cells were also strongly filipin positive (Figure 4A), indicating that these compartments contained ample free cholesterol. Unexpectedly, electron microscopy also revealed that the LEs were often in close proximity to mitochondria. Indeed, quantification revealed an increased number of contacts between mitochondria and LEs in FTY720 treated cells (Figure 4C).
Cellular Itineraries of Fluorescently labeled FTY720
We next studied the localization of FTY720 by using a fluorescently labeled FTY720. BODIPY-FTY720 reduced cholesterol esterification similarly as the nonfluorescent compound (Figure 5A). Most of the cellular BODIPY-FTY720 was nonphosphorylated, whereas the phosphorylated form predominated in the medium (Figure 5B). This is in line with the low concentrations of S1P in the cell and high concentrations in the extracellular milieu.3 In living macrophages, BODIPY-FTY720 was localized in perinuclearly concentrated tubular structures (Figure 5C). These structures became more punctuate on acLDL loading and colocalized with endocytosed DiI-labeled acLDL (Figure 5D), indicating that they represent LEs involved in the processing of modified lipoproteins.
Given the upregulation of NPC1 (but not NPC2) in FTY720-treated cells (Figure 3) and the recent suggestion that NPC1 may function as a sphingosine transporter,23 we considered that NPC1 might transport FTY720 out of the LEs. Therefore, we compared the ability of wild-type and NPC1-deficient CHO cells (M12 cells) to transport BODIPY-FTY720. We have shown that BODIPY-cholesterol accumulates in dextran-positive LEs in M12 cells.24 Using this method, we found that whereas BODIPY-cholesterol accumulated in the LEs of M12 cells, BODIPY-FTY720 did not (Figure 6). This indicates that NPC1 function is not required for the exit of FTY720 from LEs.
Mechanisms Involved in the FTY720-Induced Alterations of Cholesterol Transport
To investigate whether phosphorylation of FTY720 was necessary for the observed alterations in cellular cholesterol handling, we treated macrophages with S1P, the natural equivalent of FTY720-P. This did not reduce cholesterol esterification or increase the cellular levels of ABCA1 (Figure 7A). Furthermore, FTY720 was able to inhibit cholesterol esterification in the presence of the sphingosine kinase inhibitor N,N-dimethylsphingosine (data not shown). Finally, knockdown of the S1P1 receptor did not affect the ability of FTY720 to reduce cholesterol esterification or increase ABCA1 expression (Figure 7B). These data suggest that generation of FTY720-P and modulation of S1P receptors are not necessary for the observed effects on macrophage cholesterol balance.
ABCA1 expression is transcriptionally regulated by liver X receptor (LXR)/retinoid X receptor (RXR) heterodimers that are activated by oxysterols. We found that FTY720 increased the ABCA1 mRNA levels (36±6%, P<0.05, quadruplicate measurements from three donors). We therefore measured oxysterol levels in macrophages on FTY720 treatment. FTY720 induced a significant increase in cellular 27-hydroxycholesterol, an endogenous LXR ligand, whereas the levels of another oxysterol, 25-hydroxycholesterol, remained unaltered (Figure 7C). This indicates that FTY720 exerts a potent but selective effect on oxysterol generation.
Next, we treated macrophages either with FTY720 alone or in combination with the RXR antagonist UVI3003.25 The FTY720-induced increase in ABCA1 was abrogated by UVI3003, indicating that ABCA1 upregulation on FTY720 administration indeed occurs via nuclear hormone receptor activation (Figure 8A). When compared with a synthetic LXR ligand, T0901317, FTY720 was a weaker LXR activator, as assessed by ABCA1 upregulation. Importantly, FTY720 did not further enhance the stimulatory effect of T0901317 on ABCA1 expression (Figure 8A), suggesting that they act in the same pathway. These findings provide evidence that FTY720 stimulates 27-hydroxycholesterol formation, thereby enhancing LXR/RXR-dependent transcriptional activation and ABCA1 expression.
NPC1 expression is known to be important for the generation of 27-hydroxycholesterol in macrophages.26,27 We therefore depleted NPC1 in human primary macrophages using small interfering (si)RNAs. In NPC1-silenced cells, the stimulation of ABCA1 expression on FTY720 treatment was not observed (Figure 8B). This suggests that NPC1 plays a critical role in the process by which FTY720 stimulates 27-hydroxycholesterol production and ABCA1 upregulation. The targeting of ABCA1 to the plasma membrane is important for its function.20 FTY720 significantly increased ABCA1 levels in the plasma membrane as assessed by surface biotinylation (Figure 8C).
We show here that FTY720 reduces cholesterol deposition in human macrophage foam cells and uncover a mechanism by which FTY720 regulates sterol balance. The concentration of FTY720 producing these effects was similar to that found in target tissues during FTY720 treatment.28 Moreover, the results were surprisingly consistent in the cells of the more than 40 blood donors investigated. Although the in vivo situation is clearly much more complex, the prediction from these in vitro findings would be that FTY720 does not increase but would rather decrease the lipid burden in preexisting human atheromatous lesions.
The FTY720 induced cholesterol reduction resulted from a significant rerouting of intracellular cholesterol. This rerouting appeared distinct from that induced by other amphiphiles known to modulate the membrane activity of cholesterol (see Table I in the Online Data Supplement, available at http://circres.ahajournals.org). We found that on FTY720 treatment, endocytosed lipoprotein cholesterol was shunted less avidly to the ER for esterification and more efficiently to the extracellular acceptor apoA-I. This was observed by following the itinerary of radiolabeled cholesterol introduced in acLDL. It was also reflected in the cellular cholesterol mass, with an increase in the free versus esterified cholesterol pool and an absolute cholesterol reduction on acLDL loading in FTY720-treated cells.
Despite the relative increase in free cholesterol, FTY720 protected macrophages from free cholesterol induced cytotoxicity. This is likely to result from the FTY720-induced reduction of cholesterol transport to the ER, because reduced cholesterol delivery to the ER has been shown to protect macrophages from apoptosis.29,30 Filipin staining demonstrated that most of the free cholesterol was localized in the LEs, and from there it apparently became transferred to apoA-I. ABCA1 plays a key role in the removal of endosomal cholesterol to apoA-I.20,31 The increased ABCA1 levels on FTY720 treatment thus fit well with the enhanced cholesterol efflux to apoA-I (and a parallel decreased delivery of cholesterol to the ER). Of note, FTY720 increased ABCA1 levels and decreased the cholesterol content of macrophages also in the absence of acLDL, indicating that the process does not necessitate an excess of cholesterol.
The immunomodulatory effects of FTY720 are considered to largely derive from the modulation of S1P receptors via FTY720-P.3,4 However, we found that S1P failed to reduce cholesterol esterification and increase ABCA1 levels. In addition, cholesterol esterification was reduced under conditions where FTY720 phosphorylation was inhibited and knockdown of the receptor S1P1 did not affect the FTY720-induced reduction of cholesterol esterification or ABCA1 regulation. These results argued for an alternative mechanism of action for FTY720. As increased ABCA1 levels appeared to represent an important mediator in the FTY720-induced sterol changes, and ABCA1 is upregulated by oxysterols via LXR, we investigated whether FTY720 directly regulates oxysterol generation. We found that FTY720 induced a significant increase in 27-hydroxycholesterol production and ABCA1 transcriptional activation. To our knowledge, these results provide the first demonstration that FTY720 affects oxysterol generation and signaling.
27-Hydroxycholesterol is the most abundant circulating oxysterol and is produced by sterol-27 hydroxylase (CYP27) in several tissues. This enzyme is localized in the inner mitochondrial membrane where the cholesterol content is very low. The rate-limiting step for 27-hydroxycholesterol synthesis is considered to be the delivery of cholesterol into mitochondria.32 With this in mind, it is tempting to speculate that the increased LE–mitochondrial contacts observed on FTY720 treatment may assist in the movement of cholesterol from the LEs into mitochondria.
Moreover, NPC1 activity is known to be important for the generation of 27-hydroxycholesterol in macrophages,26,27 and our data implicate a functional role for NPC1, because the FTY720-induced ABCA1 upregulation was absent in NPC1 silenced cells. Given the sphingolipid structure of FTY720 and the LE targeting of the fluorescently labeled compound, it seems plausible that FTY720 exhibits membrane effects promoting an increase in NPC1 levels. This, in turn, might lead to increased delivery of cholesterol for mitochondrial 27-hydroxycholesterol production, possibly via increased LE–mitochondrial contacts, resulting in elevated LXR activation and ABCA1 expression (see Figure 8D).
In conclusion, this study shows that FTY720 confers atheroprotective effects in human macrophage foam cells by inhibiting the delivery of cholesterol to the ER and promoting its removal to physiological acceptors. These effects are not mediated by S1P receptor activation but rather by FTY720-induced production of 27-hydroxycholesterol, consequent LXR activation and ABCA1 upregulation. The findings indicate 27-hydroxycholesterol generation as a novel mechanism of action for FTY720. This encourages the assessment of circulating 27-hydroxycholesterol levels in human patients receiving FTY720 treatment and evaluation of the potential role of oxysterol(s) in the therapeutic actions of FTY720.
Sources of Funding
This study was supported by the Academy of Finland (grant nos. 210969 and 123261), European Union Framework Programme 7 (LipidomicNet, grant no. 202272), NIH grant HL-083187, and the Finnish Foundation for Cardiovascular Research and The Perklén Foundation. The Wihuri Research Institute is maintained by The Jenny and Antti Wihuri Foundation.
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Novelty and Significance
What Is Known?
The sphingolipid FTY720 is in clinical trials for treating immunologic disorders and acts via sphingosine-1-phosphate receptors.
Cellular sphingolipid and cholesterol metabolism are closely connected.
Whether FTY720 affects cholesterol deposition in human cells or tissues is not known.
What New Information Does This Article Contribute?
FTY720 stimulates cholesterol removal and decreases cholesterol toxicity in human macrophage foam cells.
These effects are not mediated via sphingosine-1-phosphate receptors.
Instead, we identify a novel mechanism of action for FTY720: it stimulates 27-hydroxycholesterol production, and leads to a liver X receptor dependent upregulation of the cholesterol efflux protein ABCA1.
FTY720 is a synthetic sphingolipid currently in clinical trials as an immunologic drug, eg, for multiple sclerosis. It acts by regulating sphingosine-1-phosphate receptors in lymphocytes. On a Western diet, atherosclerotic lesions are present from childhood; therefore, they are likely to be present also in individuals receiving FTY720 therapy. Sphingolipids are known to modify cholesterol metabolism. Therefore, we wondered whether FTY720 might affect atherosclerotic lesion development. Because this process is not easy to monitor in living humans, we studied monocyte-derived macrophages that represent a key cell type in atherosclerotic lesions. We found that FTY720-treated macrophages removed cholesterol better than control cells and showed increased resistance to cholesterol cytotoxicity. These effects were not mediated by sphingosine-1-phosphate receptors. Instead, we found a new mechanism that can explain the atheroprotective effects: FTY720 stimulated the production of an oxygenated cholesterol derivative, 27-hydroxycholesterol, which acts as a transcriptional activator, increasing the expression of proteins involved in cholesterol removal. These data are the first to document the effects of FTY720 on human lipid-laden macrophages (foam cells). The study identifies a novel mechanism of action for FTY720 and provides a rationale for investigating whether FTY720 increases 27-hydroxycholesterol formation and exhibits atheroprotective effects in patients receiving FTY720 therapy.
Original received July 14, 2009; revision received December 21, 2009; accepted December 23, 2009.