Download The Sphingosine-1-Phosphate Analogue FTY720 Reduces

The Sphingosine-1-Phosphate Analogue FTY720 Reduces
Atherosclerosis in Apolipoprotein E–Deficient Mice
Petra Keul, Markus Tölle, Susann Lucke, Karin von Wnuck Lipinski, Gerd Heusch,
Mirjam Schuchardt, Markus van der Giet, Bodo Levkau
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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 S1P3 sphingolipid receptor as FTY720 had no effect on thrombin-induced monocyte
chemoattractant protein-1 release in S1P3⫺/⫺ 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. (Arterioscler Thromb Vasc Biol. 2007;27:607-613.)
Key Words: sphingosine-1-phosphate 䡲 atherosclerosis 䡲 MCP-1 䡲 FTY720 䡲 ApoE⫺/⫺
T
atherosclerosis implying a TH1-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.
he 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 S1P1,-3,-4
and -53. 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 S1P1
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
(TH1)-phenotype that induce the expression of numerous
cytokines important in lesion progression and destabilisation.8 Their genetic deletion has been shown to attenuate
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.
Original received August 23, 2006; final version accepted November 27, 2006.
From the 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.
Correspondence to Bodo Levkau, Institute of Pathophysiology, University Hospital Essen, Hufelandstrasse 55, 45122 Essen, Germany. E-mail
[email protected]
© 2007 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
607
DOI: 10.1161/01.ATV.0000254679.42583.88
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Arterioscler Thromb Vasc Biol.
March 2007
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-␮m sections,
and, beginning from a random start site within the first 75 ␮m, a
section was stained every 75 ␮m 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) ⫻ (distance;
75 ␮m)].12 The aortic root was embedded in paraffin and both lesion
area and vessel area were calculated from 4 consecutive 5-␮m
sections taken every 40 ␮m 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) ⫻ (distance; 40 ␮m)]
divided by the volume of the vessel [⌺ (vessel area) ⫻ (distance;
40 ␮m)] as described.13 All analyses were performed without
knowledge of the tissue source.
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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 ␮m). 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 ␮g
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 ␮mol/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
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-␮m
sections and stained with H&E. Atherosclerotic lesion volume
was calculated from the plaque area measured in every 15th
section and the distance between sections using Cavalieri’s stereologic method [⌺ (lesion area) ⫻
(distance; 75 ␮m)].
“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
ApoEⴚ/ⴚ 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-␮m and determined lesion area at 75-␮m 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 mm3 compared with 0.11⫾0.02 mm3 in
controls, P⬍0.05) and 45.7% in female mice (0.03⫾
0.01 mm3 compared with 0.06⫾0.01 mm3 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 mm3 in FTY-treated mice compared with 0.36⫾0.04 mm3 in controls, P⬍0.05; Figure 2).
To evaluate lesion composition in both groups, we immunostained lesions for macrophages, SMCs, lymphocytes as
Keul et al
FTY720 Inhibits Atherosclerosis in ApoEⴚ/ⴚ Mice
609
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Figure 2. FTY720 reduces atherosclerotic lesions in the aortic
root. The aortic root was sectioned in 5-␮m 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.
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/mm3 in FTY-treated mice compared with
11.3⫾2.04 cells/mm3 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.
FTY720 Inhibits Thrombin-Induced Release of
Monocyte Chemoattractant Protein-1 in Aortic
Segments and VSMC
As the lesional monocyte/macrophage content in FTY720treated mice was dramatically reduced, we examined whether
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 ␮m were immunostained and the
area calculated as percent of total lesion area or cells/mm3.
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 thrombininduced 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 5A). Coincubation with the phosphorylated, biologically active form of
FTY720 (FTY720P) dose-dependently inhibited the MCP-1
release (Figure 5A). 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
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).
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Figure 5. Inhibition of thrombin-induced MCP-1 release and gene expression by FTY720, role of S1P3 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 S1P3⫺/⫺ 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 ␮mol/L) in PE-precontracted (1 ␮mol/L) aortic
rings from untreated (F) and FTY720-treated (E) C57BL/6 mice (n⫽6). Values are shown as mean⫾SEM.
stimulation was potently inhibited by FTY720P with an IC50
of ⫺8.4⫾0.2 log mol/L (Figure 5B). Using aortae from mice
deficient for the S1P3 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 5C).
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).
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⫻103/mm3 versus
2.0⫾0.3⫻103/mm3) 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 S1P1 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 longterm 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
FTY720 Inhibits Atherosclerosis in ApoEⴚ/ⴚ Mice
Keul et al
611
Cytokine Gene Expression in Thioglycolate-Elicited Peritoneal Macrophages From Mice Treated
With or Without FTY720
Gene
IL-12
IL-10
IL-4
IFN␥
Sample
⌬CT
⌬⌬CT
(⌬CT-⌬CTcontrol)
Gene Expression
FTY720 Compared to Control
(n-fold or 2⫺⌬⌬CT)
control
13.7⫾1.5
0
FTY720
13.5⫾1.3
⫺0.2
control
13.9⫾0.9
0
1
FTY720
14.3⫾0.4
0.4
0.75
1
P Value
1
1.15
control
6.3⫾0.7
0
FTY720
6.4⫾0.3
0.1
0.93
control
16.4⫾0.8
0
1
FTY720
15.8⫾1.2
⫺0.6
1.5
n.s.
n.s.
n.s.
n.s.
Real time PCR for different cytokines was performed using RNA purified from thioglycolate-elicited peritoneal
macrophages isolated from control mice (n⫽5) or mice treated with FTY720 for 4 weeks (n⫽6). Data are expressed
as n-fold differences in gene expression (2⫺⌬⌬CT) vs control mice.
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responded to FTY720P (100 pmol/L-10 ␮mol/L) with a
dose-dependent vasodilation (Figure 5D). 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
(EC50: ⫺7.2⫾0.2 log mol/L) and ⫺50.87% in controls (EC50:
⫺7.1⫾0.1 log mol/L; Figure 5D). 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 EC50: ⫺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 S1P1 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 S1P1 deficiency.6,17,18 This incomplete
lymphopenia together with the preserved functional properties of the remaining lymphocytes19 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 TH2 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 TH2-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
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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 S1P310, 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
S1P3 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 (S1P1,2,5 but not S1P3,4) 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
S1P3-mediated signaling in the vessel wall in contrast to the
loss of S1P1-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
S1P1 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 S1P1 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 receptorspecific agonists of S1P receptors that allow exploiting the
beneficial effects of FTY720 on the vasculature but avoid the
drawback of immunosuppression. Vice versa, FTY720treated 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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The Sphingosine-1-Phosphate Analogue FTY720 Reduces Atherosclerosis in
Apolipoprotein E−Deficient Mice
Petra Keul, Markus Tölle, Susann Lucke, Karin von Wnuck Lipinski, Gerd Heusch, Mirjam
Schuchardt, Markus van der Giet and Bodo Levkau
Arterioscler Thromb Vasc Biol. 2007;27:607-613; originally published online December 7,
2006;
doi: 10.1161/01.ATV.0000254679.42583.88
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Blood parameter of ApoE-/- mice after FTY720 treatment for 21 weeks.
Control (n=8)
FTY (n=9)
3.2 ± 0.48
1.9 ± 0.22
< 0.05
RBC 10 /mm
8.4 ± 0.22
8.5 ± 0.42
n.s.
HGB g/dl
11.1 ± 0.27
11.4 ± 0.65
n.s.
40.5 ± 1.07
41.2 ± 2.39
n.s.
1153.9 ± 120.26
937.0 ± 13
n.s.
2.0 ± 0.30
0.9 ± 0.13
< 0.05
MO 10 /mm
0.1 ± 0.02
0.1 ± 0.02
n.s.
GRA 103/mm3
1.1 ± 0.19
1.0 ± 0.08
n.s.
Total cholesterol mg/dl
685.9 ± 101.83
557.6 ± 104.23
n.s.
Triglycerides mg/dl
166.6 ± 27.51
141.6 ± 21.69
n.s.
WBC 103/mm3
6
3
HTC %
3
3
PLT 10 /mm
3
3
LYM 10 /mm
3
WBC:
RBC:
HGB:
HCT:
PLT:
LYM:
MO:
GRA:
3
White blood cell count
Red blood cell count
Hemoglobin concentration
Hematocrit
Platelets
Lymphocytes
Monocytes
Granulocytes