Vascular Biology |
From the Department of Applied Genetics (M.Y., T.S., H.I., Y.Y.) and Department of Medicine (T.S., H.I.., F.N.), Tokyo Medical and Dental University; and the Cardiovascular Research Center, Massachusetts General Hospital (R.E.G., A.R.), and Vascular Research Division, Department of Pathology, Brigham and Womens Hospital, Harvard Medical School (M.A.G.), Boston, Mass.
Correspondence to Masayuki Yoshida, MD, Department of Applied Genetics, Tokyo Medical and Dental University, 1-5-45, Yushima Bldg D-621, Bunkyo-ku, Tokyo 113-8510, Japan. E-mail masamgen{at}tmd.ac.jp
| Abstract |
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Key Words: HMG-CoA reductase inhibitor adhesion molecules monocytes atherosclerosis
| Introduction |
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Recently, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), which lower the plasma level of LDL cholesterol,5 have been shown to decrease the incidence of myocardial infarction and other ischemic vascular events in hyperlipidemic and atherosclerotic individuals. Interestingly, several clinical trials have suggested that there might be additional beneficial effects of statins that are independent of their cholesterol-lowering actions.6 In fact, these compounds have recently been reported to restore endothelial function via stimulating endothelial constitutive nitric oxide synthase activity in addition to lowering serum cholesterol levels.7 Moreover, HMG-CoA reductase inhibitors have been reported to be effective in blunting the hyperadhesiveness of leukocytes to endothelium in vitro8 and in vivo,9 implicating a potential effect of statin therapy on the expression and function of adhesion molecule(s). No direct studies, however, have been performed on the effects of statins on monocyteendothelial cell adhesion under physiological flow conditions. Thus, the purpose of this study was to observe the effect of cerivastatin on monocyteendothelial cell adhesive interactions in an in vitro model that mimicked physiological flow conditions and to examine the cellular mechanisms involved in modulating monocyte adhesion.
| Methods |
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S were purchased from Molecular
Probes. Cerivastatin sodium,
(+)-3R,5S-sodium-erythro-(E)-7-[4-(4-fluorphenyl)-2,6-diisopropyl-3-yl]-3,5-dihydroxyhept-6-enoate,
was a gift from Bayer AG (Leverkusen, Germany)
and was stored as a 10-mmol/L stock solution in DMSO. Monoclonal
antibodies (mAbs) used in this study were as follows: mouse anti-CD11a
(clone 38, Ancell Corp), mouse anti-CD11b (clone 44, YLEM), mouse
anti-CD18 (clone MEM48, Southern Biotechnology
Associates Inc), mouse anti-VLA4 (clone A4-PUJ1,
Upstate Biotechnology), and mouse anti-sLx
(clone KM93, Serotec Ltd). Mouse anti-RhoA mAb
was purchased from Santa Cruz Biotechnology, Inc. Wild-type and
dominant negative mutant (Asn19RhoA) forms of RhoA cDNA constructs were
kindly provided by Dr Shinya Kuroda (Nara Institute of Technology,
Nara, Japan).
Adhesion Assay Under Laminar Flow
Apparatus Design
The parallel-plate flow chamber used in the
present study was previously described in
detail.14 15
Briefly, the chamber was composed of 2 aluminum steel plates separated
by a 200-µm-thick Silastic gasket, and the flow channel was formed by
removal of a 5x20-mm rectangular section from the gasket. Defined
levels of flow were applied to the HUVEC monolayer by drawing the
perfusion medium (D-PBS containing 0.2% human serum albumin)
through the channel with a syringe pump (model 44 Harvard
Apparatus). A plastic heating plate (Tokai Hit Co) was
mounted on the stage of an inverted microscope (IX50,
Olympus) to maintain the temperature at 37°C.
The channel flow could then be approximated as a 2D fully developed
laminar flow with a simple parabolic velocity
profile.
Experimental Application
Endothelial monolayers on coverslips
were stimulated with IL-1ß (10 U/mL) for 4 hours and positioned in
the flow chamber, which was mounted on an inverted microscope. The
monolayers were perfused for 5 minutes with perfusion medium and then
examined carefully to verify the monolayer as confluent. Then, U937
cells pretreated with cerivastatin were diluted in the perfusion medium
to 106 cells/mL. The U937 cells were drawn
through the chamber at controlled flow rates to generate calculated
wall shear stresses of 1.0 and 2.0 dyne/cm2
for 10 minutes. The entire period of perfusion was recorded on
videotape with a digital video recorder containing a time
generator. Captured images were then transferred to a PC computer for
image analysis to determine the number of rolling and adherent
U937 cells in 5 to 10 randomly selected x20 microscopic fields for
each experiment. Cells were considered to be adherent after 10 seconds
of stable contact with the monolayer. Rolling leukocytes were easily
recognized, because their velocity was much slower (up to 80 µm/s)
than that of free-flowing cells.
Flow Cytometric Analysis
U937 cells were first incubated with the indicated
primary antibodies for 45 minutes on ice and washed twice with RPMI
1640/5% FCS, then incubated with an FITC-tagged goat anti-mouse
antibody (Caltag). Fluorescence was analyzed with a
FACS Calibur
(Becton-Dickinson).
Quantitation of F-Actin in U937
Cells
The filamentous (F) actin content of U937 cells after
cerivastatin treatment was quantitated as described
previously.16 In brief,
after treatment with cerivastatin (1.0 µmol/L for 48 hours), U937
cells were washed 3 times with RPMI/1% FCS and then fixed with 3.7%
formaldehyde in D-PBS for 5 minutes at 20°C. The cells were washed 3
times and then made permeable with a buffer containing 1.4%
formaldehyde in D-PBS and 0.1% NP-40 for 90 seconds at 0°C, which
was followed by staining with FITC/phalloidin (1:20 dilution in D-PBS)
for 40 minutes at 20°C. After 3 additional washes, 100 µL methanol
was added and the F-actin content was measured with a
fluorescence plate reader with an excitation wavelength of 485
nm and an emission wavelength of 535 nm.
Assays for Rho GTPase Activity
The activity of Rho GTPase was determined by 2
independent assays.
Rho Translocation Assay
A Rho translocation assay was performed as previously
described.17 18
Briefly, 1x106 U937 cells were incubated in
a lysis buffer containing 50 mmol/L HEPES (pH 7.4), 50 mmol/L
NaCl, 1 mmol/L MgCl2, 2 mmol/L EDTA,
1 mmol/L PMSF, 10 mg/mL leupeptin, 1 mmol/L
NaVO4, and 0.1% Triton
X-100 for 5 minutes on ice. The cell lysates were centrifuged
at 15 000 rpm for 15 minutes. After the supernatant had been collected
as the cytosol fraction, the pellet was resuspended in 1% Triton X-100
in the lysis buffer and centrifuged at 15 000 rpm for 15
minutes. The supernatant was then collected as the membrane fraction.
Equal amounts (10 µg) of protein from each fraction were subjected to
SDS-polyacrylamide gel electrophoresis followed by
immunoblotting with anti-RhoA mAb. The immunoreactive
RhoA proteins were detected by an enhanced chemiluminescence kit
(Amersham Pharmacia
Biotech Inc) according to the manufacturers
protocol.
RhoA GTP Binding Assay
The membrane and cytosol proteins were isolated as
described above. Protein samples (30 µg) were suspended in a mixture
consisting of 1 µmol/L of a nonhydrolyzable
fluorescence-labeled
GTP-
S19 (BODIPY FL
GTP
S, Molecular Probes), 20 mmol/L Tris,
5 mmol/L MgCl2, 1 mmol/L EDTA, and
1 mmol/L DTT, pH 8.0, in a total volume of 100 µL. A GTP-binding
reaction was carried out for 30 minutes at 22°C. Samples were then
suspended in 300 µL of immunoprecipitation buffer containing 100
mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 5 mmol/L EDTA, 1
mmol/L PMSF, 10 mg/mL leupeptin, 1 mmol/L
NaVO4, and 1% Triton X-100. Anti-RhoA mAb (5
µg) was then added to the mixture before incubation for 16 hours at
4°C. The antibody-RhoA complexes were then incubated with 50 µL of
goat anti-murine IgGcoupled Sepharose beads (Cappel) for 2 hours at
4°C. The immune complexes were collected by
centrifugation at
12 000g for 15 minutes. The
pellets were then washed 3 times with 100 mmol/L Tris (pH 7.4),
150 mmol/L NaCl, 5 mmol/L EDTA, and 1% Triton X-100. The
resulting pellets containing the immunoprecipitated fluorescent
GTP-
Slabeled RhoA were measured by a fluorescence
reader.
Overexpression of RhoA
Three expression constructs, pEF-BOS-HA-WT-RhoA,
pEF-BOS-HA-DNRhoA, and pEF-BOS, were used in the following experiment.
All vectors used a promoter of human elongation factor-1 (EF-1) to
obtain a high level of
expression.20 The vector
contains an amino-terminal hemaglutinine epitope (HA) adjacent to the
cloning sites for detection of the target protein. For transient
transfection, THP-1 cells instead of U937 cells were used, because of
their higher transfection efficiency, by a method that used a cationic
liposome (Lipofectin, Gibco-BRL). THP-1 cells
(2x106 per well) were transfected with 20
µg of pEF-BOS-HA-WT-RhoA, pEF-BOS-HA-DNRhoA, or pEF-BOS without an
insert according to the manufacturers protocol. The transfected THP-1
cells were harvested 48 hours after transfection, and RhoA expression
was determined by immunoblot with an mAb against the
HA tag.
Statistics
Data are presented as mean±SD as indicated.
Two-tailed Students t tests
were performed with Microsoft Excel. Probability
values represent the results of these
t tests, and values of
P<0.05 were considered
statistically significant.
| Results |
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HMG-CoA Reductase Inhibitor Alters
Integrin Expression of U937 Cells
To measure the cell-surface expression profiles of
adhesion molecules after cerivastatin treatment, flow cytometric
analysis of U937 cells was carried out. As shown in
Figure 2
, downregulation of CD11a, CD18, and VLA-4 was
observed after cerivastatin treatment. In contrast, the surface
expression of CD11b was not affected by cerivastatin treatment.
Expression levels of sLx, a carbohydrate structure that binds to the
selectin family of adhesion molecules, was not changed by cerivastatin
treatment. Furthermore, the attenuated expression of integrins (CD11a,
CD18, and VLA-4) was reversed by addition of mevalonic
acid.
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HMG-CoA Reductase Inhibitor
Inhibits Actin Polymerization
It is known that HMG-CoA reductase is important in
synthesizing isopropenyl moieties; therefore, the statin group of drugs
is thought to be an effective means of inactivating signaling molecules
that require isoprenylation of CAAX sequences at their C-termini, such
as small GTP-binding proteins. We also investigated the possible
involvement of Rho, a small GTP-binding protein critically important
for cell motility and cytoskeleton organization. First, we measured the
F-actin content after cerivastatin treatment of U937 cells. U937 cells
were incubated in the presence of 1.0 µmol/L cerivastatin for 48
hours, and then the amount of F-actin was measured with FITC-labeled
phalloidin. As shown in
Figure 3
, the amount of steady-state F-actin content was
significantly decreased after statin treatment (Control, 147.7±15.9
relative fluorescence units [RFU]; cerivastatin,
52±22.51, P<0.003),
suggesting that statin interfered with F-actin polymerization. F-actin
content recovered when coincubated with mevalonic
acid.
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Cerivastatin Inhibits Membrane Translocation of
Rho GTPase in U937 Cells
Because membrane translocation of Rho GTPases from the
cytosol is necessary for their proper function, we also studied the
effect of cerivastatin on membrane translocation of Rho GTPases in U937
cells. Western blotting analyses of cytosol and membrane
fractions prepared from U937 cells revealed that cerivastatin treatment
(1.0 µmol/L, 48 hours) significantly decreased the amount of
membrane-associated RhoA proteins compared with the basal condition
(Figure 4A
). Addition of mevalonic acid restored the
distribution of cytosol- and membrane-associated immunoreactive RhoA
proteins to basal levels. The level of G3PDH did not change after
statin treatment, validating the specificity of the
inhibitory effect of statin in translocation of
RhoA.
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Effect of Cerivastatin on RhoA GTP-Binding
Activity
To examine whether cerivastatin treatment affects RhoA
activity, the GTP-binding capacity of RhoA was measured in U937 cells.
Fluorescent GTP-
Sbound RhoA was immunoprecipitated from
the membrane, and the cytosol from U937 cells was treated with
cerivastatin (1.0 µmol/L) in the presence or absence of mevalonic
acid. As shown in
Figure 4B
, RhoA activity under basal conditions was 957±156
RFU in the membrane fraction and 176.3±64.5 RFU in the cytosol
fraction. Cerivastatin treatment reduced membrane-associated GTP
binding activity significantly, by 46%
(P<0.02), which was canceled
by cotreatment with mevalonic acid. GTP-binding activities of cytosol
RhoA were not significantly affected by statin
treatment.
Overexpression of Dominant Negative RhoA
Reduces THP-1 Adhesion to Activated HUVECs
To further confirm the potential role(s) of RhoA in
leukocyte adhesion, monocytic THP-1 cells with a transient expression
of wild-type (WT-RhoA) or dominant negative (DN-RhoA) RhoA were
prepared. Western blotting analysis with anti-HA mAb revealed
comparable levels of RhoA expression in both WT-RhoA and
DN-RhoAtransfected THP-1 cells
(Figure 5A
). F-actin formation was significantly inhibited in
THP-1 cells transfected with DN-RhoA, but not in those with WT-RhoA
(WT-RhoA, 54±6 RFU; DN-RhoA, 20±7.2 RFU,
P<0.0005,
Figure 5B
). Moreover, transfection of DN-RhoA significantly
reduced THP-1 adhesion to activated HUVECs compared with
WT-RhoA (WT-RhoA, 15.16±2.4% adhesion; DN-RhoA, 9±3.48%,
P<0.03,
Figure 5C
).
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C3 Exoenzyme, a Specific Inhibitor
of Rho GTPase, Reduces U937 Adhesion
To further determine the potential role of Rho GTPase
in this phenomenon, we treated U937 cells with the bacterial exoenzyme
Clostridium botulinum C3
ADP-ribosyltransferase for 48 hours at a concentration of 30 µg/mL.
This enzyme has been shown specifically to ADP-ribosylate, and thus
inactivate, Rho proteins. As shown in
Figure 6
, treatment with C3 exoenzyme for 48 hours reduced
the number of adhered U937 cells
(P<0.01 versus control U937),
which was comparable to those treated with 1.0 µmol/L cerivastatin
(P<0.003 versus control U937)
under flow conditions (shear stress=1.0
dyne/cm2).
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| Discussion |
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In the present study, we demonstrated that treatment of
a monocytic cell line, U937, with an HMG-CoA reductase
inhibitor, cerivastatin, significantly reduced adhesion to
resting and activated HUVECs under
physiological flow conditions. This effect of
cerivastatin on monocyte adhesion was concentration-dependent and was
abrogated by simultaneous treatment with mevalonic acid.
Interestingly, statin treatment did not significantly change the
fraction of rolling U937 cells under flow. One possible explanation is
that although U937 cells are able to roll on a monolayer, they
subsequently detach from the monolayer because of reduced adhesion,
rather than remain rolling on the monolayer. This suggests that the
primary target of statin action is the stable adhesion step that
follows initial rolling on activated
endothelium. Flow cytometric analysis of U937
cells after statin treatment clearly revealed downregulation of certain
cell surface integrins (CD11a, CD18, and VLA-4), which are
heterodimeric counterreceptors known to bind to ICAM-1 and VCAM-1 on
endothelial cells. These results suggest that the
mechanisms by which statins reduce U937 adhesion involve, at least in
part, the downregulation of integrin expression in U937 cells. To what
extent these integrins are responsible for this observed U937 cell
adhesion to HUVECs, however, remains to be examined. These
inhibitory effects of statins on integrin expression are
consistent with the previous findings of another
group.8 Interestingly,
Pruefer et al23 recently
reported that administration of a statin significantly decreased
P-selectindependent leukocyteendothelial cell
interactions in rat mesenteric arteries in vivo, suggesting the
potential anti-inflammatory effects of statins. Our data, obtained from
an in vitro flow-chamber system, also suggest anti-inflammatory
effects. In addition to confirming previous observations showing an
inhibitory effect of statins on
leukocyteendothelial cell adhesion under static assay
conditions,8 our experiments
were the first to examine the component step in the
leukocyteendothelial cell adhesion cascade under
physiological flow
conditions.15 Although the
amounts of shear stress used in our study were much lower than average
unidirectional arterial shear stress, they are similar to
the mean wall shear stress that has been determined for
atherosclerosis-prone human carotid artery bifurcation
(between +4 and -4
dyne/cm2).24
Because integrin-dependent leukocyte adhesion is also modulated by
receptor affinity and/or cytoskeletal
organization,25 26
we also investigated the possible involvement of Rho family proteins.
RhoA GTPase is thought to be one of the most important molecules
involved in regulation of the cytoskeleton network. A recent study
demonstrated that statin pretreatment of U937 cells directly modulated
integrin affinity via inhibition of geranylgeranylation of RhoA
protein.27 In the
present study, we were able to document that actin cytoskeleton
organization, as reflected by filamentous actin content, as well as the
activity of RhoA, judged from membrane translocation and GTP-
S
binding, were significantly altered after statin treatment. In
addition, overexpression of DN-RhoA, but not WT-RhoA, in THP-1 cells
reduced F-actin formation and attenuated their adhesion to HUVECs,
suggesting a crucial role for RhoA in this process.
It has been reported that protein geranylgeranylation is required for integrin-dependent adhesion in leukocytes27 ; thus, it is conceivable that statin treatment may affect integrin-dependent leukocyte adhesion via inhibition of the geranylgeranylation of RhoA. Although inhibition of RhoA by its specific inhibitor, C3 toxin, did not significantly alter the expression levels of integrins in U937 cells (data not shown), RhoA may modulate the affinity of integrins without changing their expression levels. Wojciak-Stothard et al25 recently reported that RhoA is required for the clustering of adhesion molecules in endothelial cells when monocytes adhere to endothelial cells. It may be possible that statin treatment directly inhibits Rho activation and disrupts actin polymerization, which leads to failure of integrin clustering, eventually resulting in reduced adhesion to endothelial cells.
Our new finding that the initial rolling step appears to be unaltered after statin treatment is consistent with the lack of any observable effect on U937 cell-surface expression of sLx, an oligosaccharide ligand of E-selectin. Statin effects on integrin expression and function may also influence the interaction of the migrating leukocyte with the subendothelial extracellular matrix, and thus leukocyte retention in the vessel wall. Although higher than those obtained in plasma, the concentrations of cerivastatin used in this study were within the range of expected tissue levels derived from prescribed pharmacological dosages.28 It remains unclear, however, whether these findings obtained from in vitro assay can be extrapolated to an in vivo situation.
Increasing evidence from controlled clinical studies indicates that statin therapy can exert a beneficial effect in various pathological conditions, including stroke,29 organ transplantation,30 31 and osteoporosis,32 independent of its lipid-lowering action.
The present study demonstrated that statin treatment of a human monocytic cell significantly reduced its adhesion, but not rolling, on activated cultured human endothelial cells under physiological flow conditions. The mechanisms by which statin reduces U937 adhesion involve reduced expression of integrins, inhibition of Rho GTPase activity, and disruption of F-actin organization. These findings provide further mechanistic insights into the added benefits of this class of lipid-lowering agents for the treatment of atherosclerotic vascular disease.
| Acknowledgments |
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Received March 4, 2001; accepted April 23, 2001.
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