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【摘要】
Objective- The sphingosine-1-phosphate (S1P) analogue FTY720 is a potent immunosuppressive agent currently in Phase III clinical trials for kidney transplantation. FTY720 traps lymphocytes in secondary lymphoid organs thereby preventing their migration to inflammatory sites. Previously, we have identified FTY720 as a potent activator of eNOS. As both inhibition of immune responses and stimulation of eNOS may attenuate atherosclerosis, we administered FTY720 to apolipoprotein E -/- mice fed a high-cholesterol diet.
Methods and Results- FTY720 dramatically reduced atherosclerotic lesion volume (62.5%), macrophage (41.8%), and collagen content (63.5%) after 20 weeks of high-cholesterol diet. In isolated aortic segments and cultured vascular smooth muscle cell, FTY720 potently inhibited thrombin-induced release of monocyte chemoattractant protein-1. This effect was mediated by the S1P 3 sphingolipid receptor as FTY720 had no effect on thrombin-induced monocyte chemoattractant protein-1 release in S1P 3 -/- mice. In contrast to S1P receptors on lymphocytes, FTY720 did not desensitize vascular S1P receptors as arteries from FTY720-treated mice retained their vasodilator response to FTY720-phosphate.
Conclusions- We suggest that FTY720 inhibits atherosclerosis by suppressing the machinery involved in monocyte/macrophage emigration to atherosclerotic lesions. As vascular S1P receptors remained functional under FTY720 treatment, S1P agonists that selectively target the vasculature and not the immune system may be promising new drugs against atherosclerosis.
The S1P analogue FTY720 potently induces immunosuppression and activates eNOS. Therefore, we tested its effect on atherosclerosis in ApoE -/- mice. FTY720 dramatically reduced lesion volume, macrophage and collagen content and potently inhibited thrombin-induced release of MCP-1 via S1P 3. Thus FTY720 may suppress the machinery regulating monocyte/macrophage emigration to atherosclerotic lesions.
【关键词】 sphingosinephosphate atherosclerosis MCP FTY ApoE /
Introduction
The novel immunomodulator FTY720 has proven highly effective in Phase III clinical trials for prevention of kidney graft rejection. 1 FTY720 is a structural homologue of sphingosine-1-phosphate (S1P), a natural sphingolipid present at high nanomolar concentrations in serum. 2 After endogenous phosphorylation, FTY720 serves as a potent agonist of 4 of the 5 G protein-coupled sphingolipid receptors S1P 1, -3, -4 and -5 3. These receptors mediate a multitude of physiological processes such as immunity, angiogenesis, cell migration, and inflammation. 4,5 The immunosuppressive effect of FTY720 has been attributed to downregulation of lymphocyte S1P 1 thus preventing effector T-cell recirculation from lymphoid organs to peripheral sites of inflammation, the prerequisite for the adaptive immune response in the T-cell compartment. 6,7
Atherosclerosis is a chronic inflammatory disease that strongly depends on T-lymphocyte-mediated immune responses for initiation and progression. 8 Atherosclerotic plaques contain activated CD4 + T-cells of the T helper 1 (T H 1)-phenotype that induce the expression of numerous cytokines important in lesion progression and destabilisation. 8 Their genetic deletion has been shown to attenuate atherosclerosis implying a T H 1-cell-driven process. 8 Although systemic administration of immunosuppressive drugs is currently not an option for treating atherosclerosis, 8 their local application in drug-eluting stents (sirolimus) has proven extremely effective in inhibiting in-stent restenosis. 9 We hypothesized that because of its immunosuppressive effect, FTY720 may have an impact on atherosclerosis. A second reason for this assumption was our recent identification of FTY720 as a potent activator of eNOS, 10 together with the evidence that NO inhibits the expression of adhesion molecules and inflammatory cytokines in atherosclerosis. 11 Therefore, we tested the effect of FTY720 on atherosclerosis in ApoE -/- mice.
Materials and Methods
Animals
FTY720 (a gift from Novartis) was administered to ApoE -/- mice in the drinking water (10 mg/L for a dose of 1.25 mg/kg/d) beginning at 4 weeks. A week after initiation of FTY720 administration, mice were started on a Western-type diet (Altromin) comprised of 21% fat by weight (0.15% cholesterol and 19.5% casein without sodium cholate) that was administered for 20 weeks.
Quantification of Atherosclerotic Lesions in the Brachiocephalic Artery and the Aortic Root
After sacrifice, mice were perfusion-fixed with 4.5% formaldehyde and the brachiocephalic artery (BCA) (from the bifurcation off the aortic arch to the branching point of the right subclavian and common carotid artery) was dissected and embedded in paraffin as described. 12 The entire BCA was serially sectioned in 5-µm sections, and, beginning from a random start site within the first 75 µm, a section was stained every 75 µm with hemalaune and erythrosine (H&E). Images were captured with a Zeiss Axio Cam, and lesion area was quantified using AxioVision 4.4 software (Carl Zeiss Vision GmbH). The volume of the BCA lesion was determined using the Cavalieri stereologic method [ (lesion area) x (distance; 75 µm)]. 12 The aortic root was embedded in paraffin and both lesion area and vessel area were calculated from 4 consecutive 5-µm sections taken every 40 µm and covering the root of the aorta; the volume fraction of the lesion was calculated as the volume of the lesion [ (lesion area including valve area) x (distance; 40 µm)] divided by the volume of the vessel [ (vessel area) x (distance; 40 µm)] as described. 13 All analyses were performed without knowledge of the tissue source.
Immunohistochemistry and Blood Analyses
The sections to be immunostained for macrophages, SMCs, and T-cells were chosen as described for quantification of lesion area (n=15 per individual BCA, every 75 µm). The antibodies used were: rabbit anti-human CD3, mouse anti-human smooth muscle-actin (Dako), and rat anti-mouse Mac-2 (Cedarline Laboratories). Primary antibodies were incubated for 1 hour at room temperature in 3% serum matched to the species of the secondary antibodies. Biotinylated secondary antibodies were incubated for 30 minutes followed by 45 minutes horseradish peroxidase-conjugated streptavidin and visualization with diaminobenzidine. Nuclei were counterstained with hemalaune. Blood was collected retroorbitally at the time of sacrifice. Blood cell counts and lipid profiles were determined using Scil vet abc and Reflotron (scil animal care company).
Measurement of MCP-1 Release and Gene Regulation
Aortae were isolated and deendothelialized as described, 14 and stimulated with thrombin in the presence or absence of FTY720P. MCP-1 secretion was measured in the supernatants using a Bioplex protein array system (BioRad). Dry weight of all used aortae was determined to normalize MCP-1 secretion for the amount of tissue. RNA was isolated from rat SMCs stimulated with thrombin in the presence or absence of FTY720P and real-time polymerase chain reaction (PCR) performed with gene-specific primers for rat MCP-1 and GAPDH (R&D Systems).
Macrophage RNA Preparation and Real Time PCR
Peritoneal macrophages were collected from C57BL/6 mice treated with or without FTY720 for 4 weeks on day 4 after intraperitoneal injection of aged, sterile 3% thioglycolate (Sigma). Total RNA was isolated using Qiagen RNeasy kit, and cDNA synthesized from 1 µg of total RNA with a RevertAid First Strand cDNA Synthesis kit (Fermentas). Real time PCR was performed using QuantiTect Primer Assays for mouse interleukin (IL)-4, IL-10, IL-12, and IFN, and GAPDH served for normalization.
Arterial Tension Studies
The vasodilator effect of FTY720P was evaluated in 2-mm rings of thoracic aortae from FTY720-treated and untreated C57BL/6 mice using established methodology. 10 After submaximal precontraction with 1 µmol/L phenylephrine (PE), dose-response curves of acetylcholine were performed to compare endothelial function between groups. After washing, rings were contracted with PE and effects of FTY720P assessed. All animal experiments were approved by the "Landesamt für Gesundheit, Ernährung und technische Sicherheit Berlin" ethics committee.
Statistical Analysis
All data are presented as means±SEM. Comparisons of the groups were performed by a nonparametric Mann-Whitney U test. Values of P <0.05 were regarded significant.
Results
FTY720 Inhibits Atherosclerotic Lesion Development and Alters Lesion Composition in ApoE -/- Mice
To examine the effect of FTY720 on atherosclerosis, we fed ApoE -/- mice a high cholesterol diet with or without oral FTY720 administration (1.25 mg/kg body weight/d) for 20 weeks. To compare lesion volume using the Cavalieri stereologic method, we examined the entire length of each brachiocephalic artery (BCA) from a random start site within the initial 75-µm and determined lesion area at 75-µm intervals (n=127 and n=90 sections in FTY-treated and control mice, respectively) as described. 12 At 20 weeks, FTY720 treatment dramatically reduced atherosclerotic lesion volume by 61.5% in male (0.04±0.01 mm 3 compared with 0.11±0.02 mm 3 in controls, P <0.05) and 45.7% in female mice (0.03± 0.01 mm 3 compared with 0.06±0.01 mm 3 in controls, P <0.05) ( Figure 1 ). FTY720 also reduced the atherosclerotic lesions in the aortic root: the volume fraction of the lesion was reduced by 46.3% (0.16±0.02 mm 3 in FTY-treated mice compared with 0.36±0.04 mm 3 in controls, P <0.05; Figure 2 ).
Figure 1. FTY720 reduces atherosclerotic lesion volume in ApoE -/- mice. Mice on Western-type diet were treated with or without FTY720, and atherosclerotic lesion volume was determined after 20 weeks. The entire BCA was sectioned in 5-µm sections and stained with H&E. Atherosclerotic lesion volume was calculated from the plaque area measured in every 15 th section and the distance between sections using Cavalieri?s stereologic method [ (lesion area) x (distance; 75 µm)].
Figure 2. FTY720 reduces atherosclerotic lesions in the aortic root. The aortic root was sectioned in 5-µm sections and 4 consecutive sections were stained with H&E. The volume fraction of the lesion was calculated as the volume of the lesion divided by the volume of the vessel.
To evaluate lesion composition in both groups, we immunostained lesions for macrophages, SMCs, lymphocytes as well as collagen, and elastin and calculated the percentage of lesional area occupied ( Figures 3 and 4 ). We examined a total of n=127 and n=90 sections in FTY-treated and control male mice, respectively, and observed a 41.8% reduction of macrophage content in lesions of FTY-treated mice (18.44±1.69%) compared with controls (31.70±1.88%). In contrast, SMC content did not differ significantly between the two groups (9.00±0.90% in FTY-treated mice compared with 6.43±0.67% in controls). The T-lymphocyte content of the lesions was low and indistinguishable between the groups (17.39±2.51 cells/mm 3 in FTY-treated mice compared with 11.3±2.04 cells/mm 3 in controls). Collagen content was 63.5% lower in lesions of FTY-treated mice (12.60±1.30%) compared with controls (34.58±2.06%). Altogether, treatment of ApoE -/- mice with FTY720 resulted in a dramatic reduction of lesion size, monocyte/macrophage content and collagen deposition.
Figure 3. Composition of atherosclerotic lesions in the BCA of control and FTY720-treated ApoE -/- mice. Representative stainings for H&E, macrophages, SMC, collagen (Sirius Red), and elastin (evG; elastica van Giesson).
Figure 4. Quantitative analysis of lesion composition in control and FTY720-treated ApoE -/- mice. In each mouse, 15 sections of the BCA at a distance of 75 µm were immunostained and the area calculated as percent of total lesion area or cells/mm 3.
FTY720 Inhibits Thrombin-Induced Release of Monocyte Chemoattractant Protein-1 in Aortic Segments and VSMC
As the lesional monocyte/macrophage content in FTY720-treated mice was dramatically reduced, we examined whether FTY720 may influence the regulation of crucial mediators of monocyte/macrophage recruitment. One such mediator is the chemokine monocyte chemoattractant protein-1 (MCP-1), which is instrumental in promoting emigration and transmigration of circulating monocytes to atherosclerotic lesions. 15 MCP-1 is produced by various cell types within the arterial wall including macrophages, endothelial cells, and VSMCs. Therefore, we tested the effect of FTY720 on thrombin-induced MCP-1 production and release in isolated rat arteries and rat VSMCs in vitro. Stimulation of deendothelialized rat aortae with thrombin (4U/mL) for 24 hours induced a robust secretion of MCP-1 into the supernatants ( Figure 5 A). Coincubation with the phosphorylated, biologically active form of FTY720 (FTY720P) dose-dependently inhibited the MCP-1 release ( Figure 5 A). In cultured rat VSMCs, FTY720P also inhibited thrombin-induced MCP-1 release (data not shown) as well as MCP-1 mRNA expression levels: the 12-fold induction of MCP-1 mRNA after 4 hours of thrombin stimulation was potently inhibited by FTY720P with an IC 50 of -8.4±0.2 log mol/L ( Figure 5 B). Using aortae from mice deficient for the S1P 3 sphingolipid receptor, we identified it as responsible for mediating the effect of FTY720P: in these, FTY720P did not attenuate thrombin-induced MCP-1 secretion while it still inhibited MCP-1 release in wild-type controls ( Figure 5 C).
Figure 5. Inhibition of thrombin-induced MCP-1 release and gene expression by FTY720, role of S1P 3 and vasodilator response to FTY720P in FTY720-treated mice. A, MCP-1 release from rat aortic segments in the supernatants after stimulation with 4U/mL thrombin for 24 hours in the presence of different concentrations of FTY720P. * P <0.05 vs thrombin. B, MCP-1 mRNA expression in rat VSMCs after stimulation with 4U/mL thrombin for 4 hours in the presence of different concentrations of FTY720P. * P <0.05 vs thrombin. C, MCP-1 release from aortic segments of C57BL/6 control mice and S1P 3 -/- mice after stimulation with 4U/mL thrombin in the presence and absence of 100 nmol/L FTY720P (n=6). D, Dose-response curves of the vasodilator effect of FTY720P (100 pmol/L-10 µmol/L) in PE-precontracted (1 µmol/L) aortic rings from untreated (-) and FTY720-treated ( ) C57BL/6 mice (n=6). Values are shown as mean±SEM.
Macrophage Cytokine Gene Expression Is Not Altered by Chronic FTY720 Treatment
FTY720 has been shown to affect major cell functions of dendritic cells and to alter their production of the cytokines IL-10 and IL-12. 16 To test whether FTY720 may also be affecting the expression of these cytokines in macrophages, we purified total RNA from thioglycolate-elicited peritoneal macrophages from mice treated with or without FTY720 for 4 weeks. Real time PCR did not reveal any differences in IL-10 or IL-12 gene expression ( Table ). The expression of two other important cytokines, IL-4 and IFN, in elicited peritoneal macrophages was not affected either ( Table ).
Cytokine Gene Expression in Thioglycolate-Elicited Peritoneal Macrophages From Mice Treated With or Without FTY720
Chronic FTY720 Treatment Impairs S1P Receptor Signaling on Lymphocytes but Preserves It in the Vasculature
Chronic FTY720 treatment significantly reduced peripheral blood lymphocyte counts by 56.6% in FTY-treated mice compared with controls (0.9±0.13 x 10 3 /mm 3 versus 2.0±0.3 x 10 3 /mm 3 ) without an effect on circulating levels of other blood cells or cholesterol levels (please see supplemental materials, available online at http://atvb.ahajournals.org). Lymphopenia attributable to downregulation and desensitization of lymphocyte S1P 1 appears to be the mechanism made responsible for immunosuppression after exposure to FTY720. 6,7 However, no data exist on the regulation and functionality of S1P receptors in the vasculature after long-term FTY720 treatment in vivo. This is particularly important as a putative downregulation and desensitization of the mechanistic action of FTY720 on vascular S1P receptors may impair its vasodilator and NO-releasing properties. To address this, we examined the vasodilator response of isolated aortae from FTY720-treated C57BL/6 mice to FTY720P. Phenylephrine (PE)-precontracted aortae from control mice responded to FTY720P (100 pmol/L-10 µmol/L) with a dose-dependent vasodilation ( Figure 5 D). Interestingly, aortae of mice chronically treated with FTY720 exhibited a similar vasodilator response to FTY720P as the controls: maximal dilatation was -53.26% in FTY720-treated mice (EC 50 : -7.2±0.2 log mol/L) and -50.87% in controls (EC 50 : -7.1±0.1 log mol/L; Figure 5 D). Thus the vasodilator response of the arterial wall to FTY720P remained functional after chronic FTY720 treatment. Vasodilation in response to acetylcholine in aortae from FTY720-treated mice was unaffected (maximal dilatation: -94.21% in FTY720-treted mice versus -90.90% in controls, and EC 50 : -7.3±0.1 log mol/L in FTY720-treated mice versus -7.2±0.1 log mol/L in controls; data not shown).
Discussion
In its function as an immunosuppressant, FTY720 has been shown to efficiently inhibit graft rejection in experimental animal models and kidney transplant patients. 7 Our finding that FTY720 inhibited atherosclerosis is in line with the important role that immunologic processes in general and T-lymphocytes in particular play in the pathogenesis of atherosclerosis. 8 The atheroprotective effect of the drug could be plausibly explained by an impairment of the adaptive immune response to atherosclerosis-related stimuli. Immunosuppression by FTY720 has been attributed to the inability of T-lymphocytes to recirculate from lymphoid organs to peripheral sites of inflammation attributable to downregulation of their S1P 1 receptors which is mirrored by the reduction of peripheral lymphocyte numbers. We have also observed a clear but incomplete lymphopenia after 21 weeks of FTY720 treatment, the longest published administration of FTY720 in mice so far. A recent study on lymphocyte kinetics during 3 weeks of FTY720 treatment supports our data by describing a 80% reduction of blood lymphocytes. 17 FTY720-induced lymphopenia has never been complete in any study so far in contrast to the virtual disappearance of lymphocytes in states of leukocyte-specific S1P 1 deficiency. 6,17,18 This incomplete lymphopenia together with the preserved functional properties of the remaining lymphocytes 19 may be an important property of FTY720 allowing residual functionality of the immune system under chronic treatment in animals and patients.
The dramatic reduction of the inflammatory burden represented by reduced macrophage content in atherosclerotic lesions of FTY720-treated mice may be a consequence of immunosuppression: An impaired immunologic response may result in defects in T-cell/macrophage communication as well as compromised antigen processing and presentation. As FTY720 has been shown to bias T cell immune responses toward T H 2 by suppressing major dendritic cell effector functions, 16 this may not only play a role in allograft-induced immune responses but also in atherosclerosis, where T H 2-cell responses have been implicated in atheroprotection. 8 Furthermore, FTY720 has also potent effects on the endothelium, and has been shown to strengthen endothelial adherens junctions and decrease VEGF-induced vascular permeability in vitro and in vivo. 20 This increase in endothelial barrier integrity may not only alter monocyte and lymphocyte trafficking but also suppress monocyte/macrophage recruitment at inflammation sites as shown in inflammatory lung injury. 21 However, a crucial component of monocyte recruitment such as the expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule (VCAM)-1 was not altered in lesions of mice treated with or without FTY720 (data not shown). Neither was the cytokine gene expression of IL-4, IL-10, IL-12, and IFN changed in thioglycolate-elicited peritoneal macrophages of FTY720-treated mice.
We suggest another mechanism of inhibition of monocyte recruitment by FTY720: inhibition of agonist-induced MCP-1 release. This may suppress the machinery involved in emigration of monocyte/macrophages to atherosclerotic lesions as MCP-1 crucially regulates monocyte/macrophage recruitment to sites of inflammation, promotes the production of reactive oxygen species, and induces the expression of proinflammatory genes in monocytes. 15 Knockout mice for MCP-1 or its receptor CCR2 have reduced atherosclerotic lesion size and macrophage recruitment. 15 This recruitment is crucially dependent on monocyte adhesion, and this may be affected by FTY720 similar to S1P, which has been shown to inhibit tumor necrosis factor (TNF) -mediated monocyte adhesion to aortic endothelium in mice. 22 We have recently provided evidence that S1P-mediated antiadhesive effects are conveyed by newly generated nitric oxide (NO): S1P inhibited both macrophage adhesion to TNF -activated endothelial cells under flow in vitro and leukocyte extravasation during myocardial ischemia/reperfusion in vivo via NO. 23 Interestingly, S1P did not inhibit macrophage rolling, but did block their firm arrest, a process known to depend on MCP-1-mediated activation of monocyte integrins. 24 MCP-1 expression, on the other hand, can be potently inhibited by NO, which has been implied in atheroprotection. 25 We have previously shown that FTY720 potently induces NO generation via S1P 3 10, and show in the present study that the same receptor mediates inhibition of MCP-1 release by FTY720. In addition, FTY720 may be improving endothelial dysfunction although eNOS mRNA and protein levels were not increased (data not shown). We did not observe any statistically significant differences in MCP-1 expression in atherosclerotic lesions of control and FTY720-treated mice (data not shown). This may have several reasons, among which probably the most important one is that the lesions we have examined were advanced, developed lesions (20 weeks) in which MCP-1 is known to be predominantly expressed by macrophages and not by endothelial of SMCs. Furthermore, MCP-1 is known to be upregulated very early (already during the first 4 weeks of high cholesterol diet in ApoE -/- mice) with levels remaining stable or even decreasing thereafter. 26 Thus the age of the lesion and its cellular composition as well as the expression kinetics of MCP-1 in vivo may be concealing the downregulation we have observed in vitro. In summary, our data support a scenario where stimulation of the S1P 3 receptor by FTY720 may protect against atherosclerosis through suppression of monocyte/macrophage recruitment.
A major characteristic of FTY720 is its selective desensitization of S1P receptors (S1P 1,2,5 but not S1P 3,4 ) through internalization. 6,27 The intact vasodilator response of aortae from FTY720-treated mice in response to FTY720P and acetylcholine suggests that there is no downregulation of S1P 3 -mediated signaling in the vessel wall in contrast to the loss of S1P 1 -mediated signaling on lymphocytes. No data exist so far on differential tissue regulation of S1P receptors by FTY720 in vivo. Although the selective downregulation of S1P receptors by FTY720 described for transfected cells holds true for lymphocytes in vivo, the situation may be very different in other cell types where internalization, recovery, or downmodulation of S1P receptors are regulated by distinct posttranslational modification pathways. 28,29 There are data in support of differences in S1P receptor regulation among tissues: treatment of endothelial cells by either FTY720P or S1P leads to a similar enhancement in barrier integrity and blockade of thrombin-induced leakage, 20,30 suggesting that S1P 1 desensitization may not occur in the endothelium as it does in lymphocytes. In support, endothelial cells in mesenteric lymph nodes have been shown to retain their abundant expression of cell surface and intracellular S1P 1 receptors after FTY720P treatment. 31 In our study, we have shown a preservation of the functional, S1P receptor-mediated vasodilator response to FTY720P in mice chronically treated with FTY720. This is particularly important as it suggests that circulating levels of FTY720 may continuously act on and protect the arterial wall against atherosclerosis by NO release and MCP-1 inhibition independently of and in concert with its putative suppressive effect on atherosclerosis-related immune processes.
In conclusion, our study has identified atheroprotection as a novel therapeutic property of FTY720, which may open new perspectives in the pharmacological treatment of atherosclerosis. This would require the design of novel receptor-specific agonists of S1P receptors that allow exploiting the beneficial effects of FTY720 on the vasculature but avoid the drawback of immunosuppression. Vice versa, FTY720-treated transplant patients may benefit not only from improved graft survival but also from the inhibition of graft atherosclerosis as shown experimentally for the heart. 32
Acknowledgments
We gratefully acknowledge the technical assistance of T. Geldermann, V. Brinkmann, A. Neugebauer, and S. Mersmann.
Sources of Funding
This work was supported by the Dr H.-H. Deichmann Foundation for Atherosclerosis Research and the DFG (LE940/3-1, SFB656, projects A1 and C3 and GI339/3-1).
Disclosures
None.
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作者单位：Institute of Pathophysiology (P.K., S.L., K.v.W.L., G.H., B.L.), University Hospital Essen, and the Med. Klinik IV (M.T., M.S., M.v.d.G.), Charite - Campus Benjamin Franklin, Berlin, Germany.