| COMMENTARIES |
The Sphingolipid Analogue FTY720 (Fingolimod) Interferes with Cholesterol Metabolism and Lymphocyte Homeostasis in Apolipoprotein E-Deficient Mice
Roland Klingenberg, M.D., Center for Molecular Medicine, Experimental Cardiovascular Research, Karolinska Hospital, L8:03, SE-17176 Stockholm, Sweden
Please address correspondence to:
Roland Klingenberg, M.D
Dept. of Cardiology
University Hospital Zürich
Rämistrasse 100
CH-8091 Zürich, Switzerland
E-mail: roland.klingenberg@usz.ch
Sphingolipids and FTY720
Sphingolipid signaling and metabolism have attracted considerable interest in vascular biology in the recent past. Sphingolipids colocalize with cholesterol in plasma membrane microdomains (lipid rafts) and are involved in key cellular functions such as cell proliferation and apoptosis, cell differentiation, cell migration, and immunological responses. Sphingosine constitutes the common backbone of several distinct sphingolipid species. Bioactive sphingosine-1-phosphate (S1P) promotes distinct effects via binding to G-protein coupled surface receptors (S1P1-5 receptors) comprising modulation of immune cell migration, macrophage differentiation, angiogenesis, vascular integrity, and enhancing eNOS-induced vasodilatation [1-3].
FTY720 (fingolimod) is a sphingolipid-like immunomodulatory substance that binds to four of the five known S1P receptors (S1P1, 3-5 receptors). After phosphorylation FTY720-P acts as an agonist upon short-term administration; its immunomodulatory effect on T lymphocytes, however, appears to be attributable to an antagonistic activity with prolonged exposure by specifically desensitizing (internalizing) the S1P1 receptor subtype, retaining lymphocytes in secondary lymphoid organs [4]. The most common side effects of FTY720 administration are a transient bradycardia, which was not detected in mice deficient in S1P3 receptor, and macula degeneration which has led to withdrawal of the drug from clinical trials of organ transplantation [summarized in 5]. Promising results were obtained in patients with relapsing multiple sclerosis receiving FTY720 in a safety and tolerability study [6] and the results from a phase III trial are awaited.
FTY720 and Atherosclerosis
Atherosclerosis is considered a chronic inflammatory disease elicited by lipid retention and modification in the arterial intima. Several inflammatory cell types such as macrophages and T lymphocytes have been identified as major players in the initiation and progression of arterial wall lesions [7]. Recent evidence shows that major perturbations occur with administration of a high-fat/high-cholesterol diet in commonly used mouse models of atherosclerosis encompassing a pronounced monocytosis with a massive increase of macrophages and T lymphocytes in atherosclerotic lesions [8-10]. With respect to sphingolipid-mediated effects, high-fat/high-cholesterol diet administered to human apoE3/4 targeted replacement mice was recently shown to modify the biological activity of VLDL lipoprotein particles promoting a S1P receptor-mediated anti-apoptotic activity for VLDL [11]. For those reasons it appeared of great interest to analyze the effects of FTY720 administration in a more physiological model of spontaneously developing atherosclerosis with less pronounced lipid levels (apoe-/- mice fed normal chow). We detected no change in atherosclerotic lesion size, cellular composition, and inflammatory mediator content [12] which is in contrast to two recent studies demonstrating an atheroprotective effect for FTY720 administration associated with a decreased recruitment of inflammatory cells into the atherosclerotic lesions in the LDLr-/- and apoe-/- mouse model under conditions of a high-fat/high-cholesterol diet [13,14].
Oral administration of FTY720 to apoe-/- mice on normal chow for 12 weeks mediated a 2.4-fold increase in serum cholesterol levels (predominantly VLDL-fraction). FTY720 was previously shown to inhibit S1P degradation by inhibition of the S1P lyase [15], explaining our finding of a significant increase in plasma S1P levels. In turn, sphingosine and S1P were shown to induce cholesterol biosynthesis via activation of HMGCoA reductase and SREBP1 [16,17] corroborating our finding of a 1.8-fold increase in HMGCoA reductase mRNA. This effect may have been masked by feeding a high-fat/high-cholesterol diet in the two other studies [13,14] as the diet in itself promotes a marked increase in non-HDL cholesterol levels [18].
In addition, FTY720 administration mediated peripheral blood lymphopenia (70% reduction) associated with a discoordinate redistribution of T and B lymphocytes and monocytes into secondary lymphoid organs. Lymphocyte function was unaltered as analyzed by proliferation assays and anti-oxLDL antibody titers. The overall depletion was associated with a relative (2-fold) increase in both effector memory T cell and regulatory T cell content as absolute cell numbers for both T cell subtypes remained constant. As effector memory T cells are generated upon antigen encounter in the periphery and are not derived from the thymus, our data suggest continuous renewal of this cell-type in the periphery with long-term FTY720 administration. As regulatory T cell numbers also remained constant we suggest that in the presence of FTY720 those cells are generated from effector memory cells as previously described [19] – and are not of thymic origin as FTY720 inhibits thymic egress of lymphocytes [4].
Conclusions
We report the novel finding that FTY720 administered to apoe-/- mice on normal chow altered lipid metabolism yielding pronounced hypercholesterolemia. Concomitant lymphocyte depletion and redistribution was not associated with a shift in the balance of pro- and anti-atherogenic lymphocyte populations. Hypercholesterolemia appears to have counteracted the otherwise beneficial effect on the development of atherosclerosis. It is conceivable that the difference in outcome with respect to atherosclerosis between our study [12] and the two other studies [13,14] is due to the effects of a high-fat/high-cholesterol diet fed to hyperlipidemic mice. Accordingly, a massive increase in monocyte/macrophage numbers promoting atherogenesis [8] could be switched into an anti-inflammatory phenotype [2] by the agonistic effects of the sphingosine-1-phosphate analogue FTY720 and thus attenuate the development of atherosclerosis. Our data suggest careful interpretation of anti-atherosclerotic effects of sphingolipid drugs in mouse models with altered lipid metabolism. It will be interesting to learn whether the novel side effect of hypercholesterolemia is restricted to the apoe-/- mouse model with its altered lipid clearance and whether it could be avoided by the use of more selective sphingosine-1-phosphate receptor agonists.
References