(Circulation. 2000;102:3104.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Suppression of Endothelial Nitric Oxide Production After Withdrawal of Statin Treatment Is Mediated by Negative Feedback Regulation of Rho GTPase Gene Transcription
From the Medizinische Klinik und Poliklink, Universitatskliniken des Saarlandes, Homburg, Germany (U.L, F.C., G.N., M.B.); the Neurologische Klinik der Charite, Humboldt-Universität zu Berlin, Berlin, Germany (M.E., K.G.); and the Cardiovascular Division, Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass (J.K.L.).
Correspondence to Ulrich Laufs, MD, Medizinische Klinik und Poliklink, Universitatskliniken des Saarlandes, Innere Medizin III, Homburg, 66421 Germany. E-mail Ulrich{at}Laufs.com
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background—Statins improve endothelial function by upregulating endothelial nitric oxide (NO) production that is mediated by inhibiting the isoprenylation of rho GTPase. Withdrawal of statin treatment could suppress endothelial NO production and may impair vascular function.
Methods and Results—To test this hypothesis, mice were treated for 14 days with 10 mg/kg atorvastatin per day; this led to the upregulation of endothelial NO synthase expression and activity by 2.3- and 3-fold, respectively. Withdrawal of statins resulted in a dramatic, 90% decrease of NO production after 2 days. In mouse aortas and cultured endothelial cells, statins upregulated the expression of rho GTPase in the cytosol, but statins blocked isoprenoid-dependent rho membrane translocation and GTP-binding activity. Inhibiting the downstream targets of rho showed that rho expression is controlled by a negative feedback mechanism mediated by the actin cytoskeleton. Measuring rho mRNA half-life and nuclear run-on assays demonstrated that statins or disruption of actin stress fibers increased rho gene transcription but not rho mRNA stability. Therefore, treatment with statins leads to the accumulation of nonisoprenylated rho in the cytosol. Withdrawing statin treatment restored the availability of isoprenoids and resulted in a massive membrane translocation and activation of rho, causing downregulation of endothelial NO production.
Conclusions—Withdrawal of statin therapy in normocholesterolemic mice results in a transient increase of rho activity, causing a suppression of endothelial NO production. The underlying molecular mechanism is a negative feedback regulation of rho gene transcription mediated by the actin cytoskeleton.
Key Words: nitric oxide • endothelium • statins • rho GTPase
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Large clinical trials have demonstrated that hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) reduce the risk of myocardial infarction and stroke.1 Increasing evidence suggests that statins may exert effects on the vasculature beyond the lowering of serum cholesterol levels.1 2 3 4 5 Indeed, it has been shown that HMG-CoA reductase inhibitors upregulate the expression and function of endothelial nitric oxide synthase (eNOS) independent of cholesterol levels.6 7 8 9 10 11 12 Impaired release of endothelial nitric oxide (NO) causes endothelial dysfunction. Endothelium-derived NO mediates vasodilatation and inhibits leukocyte adhesion, platelet aggregation, and smooth muscle cell proliferation. The inactivation of endothelial NO by superoxide anion leads to nitrate tolerance and vasoconstriction.13 In animal models, the upregulation of eNOS by statin treatment improved cardiac function9 and decreased stroke size during ischemia.8 12 The reduction of stroke size after statin treatment is mainly mediated by endothelial NO, because mutant mice lacking the gene for eNOS were not protected from cerebral ischemia.8
However, despite the clinical effects of statins, the effects of withdrawing statin treatment are not known. Interestingly, a recent study noted a 3-fold increase in thrombotic vascular events after the treatment of patients with simvastatin was stopped and then continued with relatively lower doses of fluvastatin because of reference pricing for statins in New Zealand.14 We hypothesized that terminating statin treatment may suppress endothelial NO production and impair vascular function.
Upregulation of endothelial NO by statins is mediated by decreased levels of geranylgeranylpyrophosphate (GGPP), an isoprenoid intermediate of the cholesterol synthesis pathway.15 Isoprenoids are important for the post-translational modification of proteins, such as the small GTP-binding proteins ras and rho.16 Isoprenylation of small GTPases is necessary for intracellular trafficking and membrane association.17 18 We identified the small G-protein rho as a negative regulator of endothelial NO release. Hence, statins increase endothelial NO production by inhibiting the geranylgeranylation of rho.12 15 Other cholesterol-independent effects of statins may also be mediated by the inhibition of rho GTPase, such as the release of tissue plasminogen activator19 or the inhibition of cell-cycle progression of vascular smooth muscle cells.20 Indeed, rho GTPases play a key role in the regulation of actin stress-fiber formation, the organization of the actin cytoskeleton, cell growth, and cell proliferation.18 21 22 23 However, despite the importance of rho for cellular function, little is known about the regulation of rho expression and activity in endothelial cells.24
We reasoned that statin-induced reduction of rho isoprenylation inhibits rho activity rather than rho production. Therefore, we hypothesized that an early withdrawal of statin therapy could lead to a profound rebound phenomenon, because the rapid isoprenylation of cytosolic rho could induce massive membrane translocation and activation of rho, leading to the suppression of eNOS activity and expression.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Materials
Cytochalasin D (cytoD), mevastatin, farnesylpyrophosphate, GGPP, L-mevalonate, and 5,6-dichlorobenzimidazole riboside (DRB) were purchased from Sigma. H-7, ML-7, 2,3-butanedione monoxime, and nocodazole were from Calbiochem. Clostridium botulinum C3 transferase was purchased from List Biological Laboratories. [32P]-dCTP, [32P]-UTP, L-[2,3,4,5-3H]arginine-monohydrochloride, Hybond N-nylon membranes, and x-ray film were obtained from Amersham. [35S]GTP
S was supplied by New
England Nuclear. Atorvastatin was obtained from Gödecke-Parke-Davis,
and simvastatin and lovastatin were a gift from Merck-Sharp-Dohme
(Haar, Germany). Simvastatin, lovastatin, and mevalonate were activated
by alkaline
hydrolysis.6
Cell Culture
Endothelial cells were harvested from bovine
aortas.25 Confluent
endothelial cells from <4 passages cultured with 10% fetal calf serum
were used for all treatment conditions. In some experiments,
endothelial cells were treated with DRB for 30 minutes before the
addition of atorvastatin and cytoD. Cellular viability was determined
by cell count, morphology, and trypan blue
exclusion.
Animal Treatment
All animal experiments were conducted in accordance
with the guidelines of the National Institutes of Health and the
authors’ institutions. Mice (strain 129/SV; weighing 18 to 22 g)
were injected subcutaneously with 0.1 mL of atorvastatin (1 or 10
mg/kg) or PBS once daily for 14 days. Animals were treated and killed
under identical external conditions (time of day, temperature, light,
etc).
eNOS Activity Assay
Endothelial cells and mouse aortas were homogenized
in 250 mmol/L Tris-HCl (pH 7.4), 10 mmol/L EDTA, and 10 mmol/L EGTA.
Lysates were pelleted (10 minutes at 13000 rpm and 4°C), and
supernatants were used for the assay. The total protein concentration
in the supernatants was quantitated, and 10 µg of protein per sample
was used. eNOS activity was determined by measuring the conversion of
[3H]-arginine to
[3H]-citrulline using the NOS assay kit
from Calbiochem. Rat cerebellum served as positive control. Lysates
incubated with the eNOS inhibitor nitro-L-arginine methyl
ester L (1 mmol/L) served as blanks. Time-course experiments
showed that basal NO production in confluent endothelial cells did not
change between 0 and 24 hours.
Western Blotting
Total cell lysates and membrane and cytosolic
proteins were isolated as described
previously.15 Immunoblotting
was performed using a rhoA monoclonal antibody (1:250 dilution; Santa
Cruz Biotechnology), donkey-anti–rabbit secondary antibody (1:4000
dilution), and the enhanced chemiluminescence kit
(Amersham).
Northern Blotting
Northern blotting using
[32P]-labeled full-length rhoA
cDNA26 was performed as
described
previously.6
Assay for Rho GTP-Binding Activity
Rho GTP-binding activity was determined by specific
immunoprecipitation of
[35S]GTPS–labeled rho, as described
previously.15 25
eNOS Reverse Transcription–Polymerase
Chain Reaction
RNA isolation, reverse transcription, and competitive
polymerase chain reaction (PCR) were performed according to standard
techniques. The sense (5'-TTCCGGCTGCCACCTGATCCTAA-3') and antisense
(5'-AACATATGTCC TTGCTCAAGGCA-3') primers were used to amplify a 340-bp
murine eNOS cDNA fragment and a 1052-bp mutated eNOS cDNA fragment,
which served as an internal standard. GAPDH was amplified as the
external standard.8 Each PCR
cycle consisted of denaturing at 94°C for 30 seconds, annealing at
60°C for 30 seconds, and elongating at 72°C for 60 seconds. The
linear exponential phases for eNOS and GAPDH PCR were 35 and 22 cycles,
respectively. Equal amounts of corresponding NOS and GAPDH reverse
transcription-PCR products were loaded on agarose gels, and optical
densities of ethidium bromide–stained DNA bands were expressed as the
ratio of murine eNOS to eNOS mutant PCR signal.
Nuclear Run-On Assays
Nuclei of endothelial cells were isolated, and in
vitro transcription was performed in the presence of
[32P]-UTP.6
Purified, denatured, full-length rhoA-, GAPDH- and linearized pcDNA3
cDNA were vacuum-transferred onto nitrocellulose membranes.
Hybridization of radiolabeled mRNA transcripts to the membranes was
performed at 45°C for 24
hours.6
Data Analysis
Band intensities were analyzed by densitometry. All
values are expressed as mean±SEM compared with controls. Paired and
unpaired Student’s t tests and
ANOVA for multiple comparisons were employed. Differences were
considered significant at
P<0.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Withdrawal of Statin Treatment Transiently Decreases eNOS Activity
The effect of atorvastatin on eNOS activity was assessed by [3H]arginine to [3H]citrulline conversion assay.8 12 Treatment of bovine aortic endothelial cells with 1, 5, and 10 µmol/L atorvastatin for 24 hours increased eNOS activity in a concentration-dependent way to 130±7%, 170±12%, and 219±8.3%, respectively, compared with control (n=5 separate experiments; P<0.05). To study the effects of terminating statin treatment, endothelial cells were treated with atorvastatin (10 µmol/L). After 24 hours, the drug was completely removed by replacing the medium, and eNOS activity was measured at the indicated time points (Figure 1
). At 8, 12, and 16 hours after stopping statin
treatment, eNOS activity decreased 142%, 43%, and 37%, respectively,
compared with untreated cells. After 20 hours, eNOS activity increased
61% and, after 24 hours, eNOS activity returned to control
levels.
|
Inhibition of Isoprenoid Synthesis Upregulates
Rho Expression
Rho GTPase negatively regulates eNOS expression
and function.15 Therefore,
the effects of atorvastatin on rho protein expression in endothelial
cells were characterized. Atorvastatin (1 µmol/L) time-dependently
(Figure 2A) and concentration-dependently (data not shown)
upregulated rho protein expression. Upregulation of rho expression by
atorvastatin was a class effect of HMG-CoA reductase inhibitors:
mevastatin, lovastatin, and simvastatin also upregulated rho expression
(data not shown). Upregulation of rho was mediated by the inhibition of
mevalonate synthesis; the addition of L-mevalonate completely reversed
the effects of the statins
(Figure 2B). Similarly, adding the isoprenoid GGPP reversed
the upregulation of rho by statins. In contrast, adding
farnesylpyrophosphate had no effect. These findings indicate
that rho expression is negatively regulated by
geranylgeranylation.
|
Rho Is Negatively Regulated by the Actin
Cytoskeleton
How rho expression in endothelial cells is regulated is
not known. Downstream targets of rho GTPase include the regulation of
myosin light chain (MLC) phosphorylation and actin stress fiber
formation.12 18 26
To test the hypothesis that rho might be negatively regulated by its
downstream effectors, we inhibited several levels of the signal
transduction cascade downstream of rho. Direct inactivation of rho
GTPase activity by ADP-ribosylation with
Clostridium botulinum C3
transferase (50 µg/mL, 24
hours)17 resulted in the
upregulation of rho protein expression
(Figure 3A). Inhibiting MLC kinase with H-7 (10 µmol/L) or
2,3-butanedione 2-monoxime time-dependently increased rho expression,
with a maximum effect after 8 hours (data not shown). Inhibiting MLC
phosphorylation and MLC ATPase prevents the formation of actin stress
fibers.26 Therefore, we
directly disrupted actin stress fibers with cytoD. Treating endothelial
cells with cytoD for 8 hours showed a dose-dependent increase of rho
protein
(Figure 3B) and mRNA expression (data not shown).
Upregulation of rho was specific to changes in microfilaments rather
than the microtubule cytoskeleton because treatment with nocodazole
slightly decreased rho expression
(Figure 3C). As a negative control, the expression of rac
GTPase was studied
(Figure 3D). Interestingly, rac was upregulated by
atorvastatin in a manner similar to that of rho, but although cytoD
upregulated rho, it had no effect on rac expression. In summary, these
data indicate that actin stress fiber formation negatively regulates
rho expression.
|
Rho function depends on its membrane-associated GTP binding
activity rather than rho expression in total cell
lysates.18 26
Therefore, the effect of disrupting actin stress fibers with cytoD (1
µmol/L, 24 hours) on rho expression in isolated cell membranes and
the cytosol was studied. Treatment with atorvastatin and simvastatin
(10 µmol/L, 24 hours) upregulated rho expression in the cytosol of
endothelial cells
(Figures 4A and 4B). Similarly, cytoD increased cytosolic rho.
Conversely, inhibiting rho isoprenylation by statins decreased the
translocation of rho to the cell membrane, but disrupting actin stress
fibers downstream of rho upregulated rho expression in the cell
membrane.
|
To determine whether rho function is regulated by statins
and the actin cytoskeleton, we immunoprecipitated
[35S]GTPS-labeled rho from cell
membranes. Treatment with atorvastatin decreased rho GTP-binding
activity by 50%
(Figure 4C
). In contrast, cytoD upregulated rho function by
25%. Taken together, these findings show that statins upregulate rho
expression but inhibit rho function, and they identify the actin
cytoskeleton as a negative regulator of rho expression and
function.
Rho Gene Transcription Is Regulated by the
Actin Cytoskeleton
To characterize the molecular regulation of rho
expression, rho mRNA half-life was determined using the RNA polymerase
inhibitor DRB. There was no significant difference in
post-transcriptional regulation of rho mRNA after the treatment of
endothelial cells with cytoD
(Figures 5A and 5B) or atorvastatin (data not
shown).
|
To determine whether the effects of atorvastatin and cytoD
on rho expression occur at the level of rho gene transcription, we
performed nuclear run-on assays using endothelial cells treated with
atorvastatin (10 µmol/L, 12 hours) or cytoD (1 µmol/L, 12 hours)
(Figure 5C). GAPDH served as the internal standard, and the
empty pcDNA3 cDNA vector was used to determine nonspecific
hybridization. Nuclear run-on assays showed that atorvastatin and cytoD
increased rho gene transcription by 3-fold (315±42% and 287±30%,
respectively; n=3 separate experiments;
P<0.05).
Termination of Statin Treatment Transiently
Increases Rho Membrane Translocation
To study the effects of stopping statin treatment on
rho expression, cytosolic and membrane proteins of endothelial cells
were analyzed. In the presence of atorvastatin, rho protein expression
decreased in the membrane but increased in the cytosol
(Figure 6). Three hours after the withdrawal of atorvastatin,
rho expression started to increase in the membrane and, after 6 hours,
rho protein expression exceeded control levels. Rho membrane expression
remained upregulated after 8 hours and then gradually returned to
baseline levels after 16 hours. In contrast, rho expression in the
cytosol was downregulated after 3 and 6 hours, suggesting a massive
translocation of rho from the cytosol to the cell membrane. Rho
expression in the cytosol increased to control levels after 16
hours.
|
Withdrawal of Statin Treatment Transiently
Decreases Endothelial NO Production In Vivo
To determine the effects of atorvastatin on eNOS in
vivo, 129/SV wild-type mice were subcutaneously injected daily for 14
days with 1 and 10 mg/kg atorvastatin. As observed previously with
simvastatin and lovastatin, serum cholesterol levels did not change
significantly when treated with 10 mg/kg atorvastatin (data not
shown).8 Compared with
control mice, treatment with 1 and 10 mg/kg atorvastatin
concentration-dependently upregulated eNOS mRNA (165±15% and
230±29%, respectively; n=8,
P<0.05 for each
group).
To determine the effect of stopping statin treatment in
vivo, mice were treated with 10 mg/kg atorvastatin. After 14 days,
treatment was stopped and aortas were harvested after 2 and 4 days. Two
days after terminating statin treatment, eNOS mRNA expression decreased
5-fold (46±7%;
Figures 7A and 7B). Four days after the discontinuation of
statin treatment, eNOS mRNA expression returned to control
levels.
|
To determine the effects of statin withdrawal on endothelial
NO production in vivo, [3H]arginine to
[3H]citrulline conversion assays were
performed. Treatment with atorvastatin resulted in a 3-fold
upregulation of NOS activity in the aorta compared with vehicle
(Figure 7C). Two days after the cessation of statin
treatment, NOS activity decreased by 10-fold (32% compared with
vehicle). Four days after stopping treatment, NOS activity returned to
baseline levels. As observed previously in mice treated with
simvastatin,8 inducible
NOS mRNA was not detected in the aortas of atorvastatin- or
vehicle-treated mice.
Termination of Statin Treatment Increases Rho
Membrane Translocation in Vivo
To determine whether statins regulate rho membrane
expression in vivo, Western analysis of membrane and cytosolic proteins
of aortas from mice treated with atorvastatin were performed.
Statin-treated mice showed upregulation of rho in the cytosol
(Figure 8). In the membrane, atorvastatin decreased rho
protein expression. Two days after terminating statin treatment, rho
membrane expression increased 3-fold but cytosolic rho expression was
downregulated. Four days after stopping statin treatment, rho
expression returned to baseline levels. These data show, for the first
time, that statins regulate vascular rho expression and localization in
vivo.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
We showed that 2 days after the withdrawal of statin treatment, endothelial NO production decreased by 90%. The molecular mechanism of this finding was identified as the regulation of the small GTP-binding protein rho. Statin treatment inhibits rho isoprenylation and function, and rho is a negative regulator of eNOS expression.15 Statins upregulate eNOS by inhibiting rho function. Surprisingly, the inhibition of endothelial isoprenoid synthesis by statins increased rho gene transcription. This effect was mediated by a negative feedback signal of the actin cytoskeleton. In the presence of statins, however, the increased amount of transcribed rho cannot be geranylgeranylated and it accumulates in the cytosol of the endothelial cell; therefore, it is functionally inactive. Withdrawing statin treatment restores the availability of GGPP. The inactive (GDP-bound) cytosolic rho is isoprenylated, translocates to the cell membrane, and becomes activated (GTP-bound). This transient increase of rho GTP-binding activity causes the decrease of endothelial NO production.
Treatment of normocholesterolemic mice with atorvastatin for 14 days increased endothelial NO production but did not alter serum cholesterol levels. Upregulation of endothelial NO has been observed with different statins in a variety of species, including humans.1 8 9 10 11 12 NO is a key mediator of vascular homeostasis and blood flow. Decreased amounts of endothelial NO impair vascular function by promoting vasoconstriction, platelet aggregation, smooth muscle cell proliferation, and leukocyte adhesion.13 Our experiments show that terminating statin treatment reverses the upregulation of eNOS and transiently decreases NO production below baseline levels.
Rho GTPases are important mediators of vascular function. For example, in addition to negatively regulating eNOS expression,15 rho controls the release of tissue plasminogen activator19 and the adhesion of monocytes to the endothelium.27 Rho mediates the proliferation and migration of vascular smooth muscle cells15 28 and is a mediator of mechanotransduction in the vascular wall.23 In cardiac myocytes, rho serves as a regulator of the signaling pathway leading to hypertrophy.21 22 Yet, the available information about the regulation of rho expression is limited. In the present study, we show that statin treatment transcriptionally upregulates rho expression but post-translationally inhibits rho membrane–associated function in the vascular wall. Rho geranylgeranylation, membrane translocation, and GTP-binding activity are inhibited in the presence of statins. Recently, several cholesterol-independent effects of statins have been reported.29 30 31 32 Inhibiting the isoprenylation of small GTP-binding proteins is an important, cholesterol-independent effect of statin treatment.12 15 19 20 Our study demonstrates that statins can be used as tools to modify rho function in vivo.
Interestingly, the direct inhibition of rho GTPase activity by C3 transferase upregulates rho expression. Similarly, inhibiting MLC phosphorylation and directly disrupting actin stress fibers lead to increased rho expression. These experiments identify the actin cytoskeleton as a downstream sensor of rho function in the endothelium. The molecular mechanism of this feedback regulation of rho expression by the actin cytoskeleton was identified as an increase in gene transcription but not rho mRNA stability. These findings agree with a recent study showing that rho activation in Swiss 3T3 cells is negatively regulated by cytoskeletal structures during adhesion to the extracellular matrix.24 Further experiments are needed to characterize the signaling from the actin cytoskeleton to the rho promoter.
Recent evidence suggests that statins exert beneficial vascular effects in addition to lowering serum cholesterol. Indeed, recent and ongoing clinical trials are comparing statin therapy with angioplasty33 and testing the effects of statins on acute coronary syndromes. In the present study, we show that terminating statin treatment suppresses endothelial NO production. Therefore, withdrawing statin therapy may acutely impair vascular function and precipitate acute coronary syndromes (eg, when statin therapy is discontinued during the early postoperative period after aortocoronary bypass surgery or when patients are switched from one statin drug to another).14 Clearly, clinical studies are needed to characterize the time course and significance of the clinical events after the withdrawal of statin therapy and their correlation with the downregulation of eNOS activity.
|
Acknowledgments |
|---|
This work was supported by the Deutsche Forschungsgemeinschaft (La 1057/4-1 to U.L.; En 343/4-1 and En 343/6-1 to M.E.), the Bundesministerium für Bildung, Forschung und Technologie (BMBF), the Köln Fortune Program, and the Hermann und Lilly Schilling Foundation. We thank I. Paez-Mallez for excellent technical assistance and A. Hall for kindly providing rhoA cDNA.
Received June 9, 2000; revision received August 3, 2000; accepted August 4, 2000.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Maron DJ, Fazio S, Linton MF. Current perspectives on statins. Circulation. 2000;101:207–213.
2.
O’Driscoll G,
Green D, Taylor RR. Simvastatin, an HMG-coenzyme A reductase inhibitor,
improves endothelial function within 1 month.
Circulation. 1997;95:1126–1131.
3.
Packard CJ.
Influence of pravastatin and plasma lipids on clinical events in the
West of Scotland Coronary Prevention Study (WOSCOPS).
Circulation. 1998;97:1440–1445.
4. Massy ZA, Keane WF, Kasiske BL. Inhibition of the mevalonate pathway: benefits beyond cholesterol reduction? Lancet. 1996;347:102–103.[Medline] [Order article via Infotrieve]
5.
Wagner AH, Kohler
T, Ruckschloss U, et al. Improvement of nitric oxide-dependent
vasodilatation by HMG-CoA reductase inhibitors through attenuation of
endothelial superoxide anion formation.
Arterioscler Thromb Vasc Biol. 2000;20:61–69.
6.
Laufs U, La Fata V,
Plutzky J, et al. Upregulation of endothelial nitric oxide synthase by
HMG CoA reductase inhibitors.
Circulation. 1998;97:1129–1135.
7. Hernandez-Perera O, Perez-Sala D, Navarro-Antolin J, et al. Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells. J Clin Invest. 1998;101:2711–2719.[Medline] [Order article via Infotrieve]
8.
Endres M, Laufs U,
Huang Z, et al. Stroke protection by 3-hydroxy-3-methylglutaryl
(HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide
synthase. Proc Natl Acad Sci
U S A. 1998;95:8880–8885.
9.
Lefer AM, Campbell
B, Shin YK, et al. Simvastatin preserves the ischemic-reperfused
myocardium in normocholesterolemic rat hearts.
Circulation. 1999;100:178–184.
10.
Kaesemeyer WH,
Caldwell RB, Huang J, et al. Pravastatin sodium activates endothelial
nitric oxide synthase independent of its cholesterol-lowering actions.
J Am Coll Cardiol. 1999;33:234–241.
11. Tannous M, Cheung R, Vignini A, et al. Atorvastatin increases ecNOS levels in human platelets of hyperlipidemic subjects. Thromb Haemost. 1999;82:1390–1394.[Medline] [Order article via Infotrieve]
12. Laufs U, Endres M, Stagliano N, et al. Neuroprotection mediated by changes in the endothelial actin cytoskeleton. J Clin Invest. 2000;106:15–24.[Medline] [Order article via Infotrieve]
13.
Liao JK.
Endothelium and acute coronary syndromes.
Clin Chem. 1998;44:1799–1808.
14. Thomas M, Mann J. Increased thrombotic vascular events after change of statin. Lancet. 1998;352:1830–1831.[Medline] [Order article via Infotrieve]
15.
Laufs U, Liao JK.
Post-transcriptional regulation of endothelial nitric oxide synthase
mRNA stability by rho GTPase. J Biol
Chem. 1998;273:24266–24271.
16. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature. 1990;343:425–430.[Medline] [Order article via Infotrieve]
17. Aktories K. Bacterial toxins that target rho proteins. J Clin Invest. 1997;99:827–829.[Medline] [Order article via Infotrieve]
18.
Van AL,
D’Souza-Schorey C. Rho GTPases and signaling networks.
Genes Dev. 1997;11:2295–2322.
19.
Essig M, Nguyen
G, Prie D, et al. 3-Hydroxy-3-methylglutaryl coenzyme A reductase
inhibitors increase fibrinolytic activity in rat aortic endothelial
cells: role of geranylgeranylation and rho proteins.
Circ Res. 1998;83:683–690.
20.
Laufs U, Marra D,
Node K, et al. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors
attenuate vascular smooth muscle proliferation by preventing rho
GTPase-induced down- regulation of p27(Kip1).
J Biol Chem. 1999;274:21926–21931.
21.
Aikawa R, Komuro
I, Yamazaki T, et al. Rho family small G proteins play critical roles
in mechanical stress- induced hypertrophic responses in cardiac
myocytes. Circ Res. 1999;84:458–466.
22. Finkel T. Myocyte hypertrophy: the long and winding RhoA’d. J Clin Invest. 1999;103:1619–1620.[Medline] [Order article via Infotrieve]
23.
Numaguchi K,
Eguchi S, Yamakawa T, et al. Mechanotransduction of rat aortic vascular
smooth muscle cells requires RhoA and intact actin filaments.
Circ Res. 1999;85:5–11.
24. Ren XD, Kiosses WB, Schwartz MA. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 1999;18:578–585.[Medline] [Order article via Infotrieve]
25.
Miyamoto A, Laufs
U, Pardo C, et al. Modulation of bradykinin receptor ligand binding
affinity and its coupled G-proteins by nitric oxide.
J Biol Chem. 1997;272:19601–19608.
26.
Hall A. Rho
GTPases and the actin cytoskeleton.
Science. 1998;279:509–514.
27.
Wojciak-Stothard
B, Williams L, Ridley AJ. Monocyte adhesion and spreading on human
endothelial cells is dependent on Rho-regulated receptor clustering.
J Cell Biol. 1999;145:1293–1307.
28.
Seasholtz
TM, Majumdar M, Kaplan DD, et al. Rho and rho kinase mediate
thrombin-stimulated vascular smooth muscle cell DNA synthesis and
migration. Circ Res. 1999;84:1186–1193.
29.
Colli S, Eligini
S, Lalli M, et al. Vastatins inhibit tissue factor in cultured human
macrophages: a novel mechanism of protection against atherothrombosis.
Arterioscler Thromb Vasc Biol. 1997;17:265–272.
30.
Pruefer D, Scalia
R, Lefer AM. Simvastatin inhibits leukocyte-endothelial cell
interactions and protects against inflammatory processes in
normocholesterolemic rats. Arterioscler
Thromb Vasc Biol. 1999;19:2894–2900.
31. Rosenson RS, Tangney CC, Casey LC. Inhibition of proinflammatory cytokine production by pravastatin. Lancet. 1999;353:983–984.[Medline] [Order article via Infotrieve]
32.
Mundy G, Garrett
R, Harris S, et al. Stimulation of bone formation in vitro and in
rodents by statins. Science. 1999;286:1946–1949.
33.
Pitt B, Waters D,
Brown WV, et al. Aggressive lipid-lowering therapy compared with
angioplasty in stable coronary artery disease: Atorvastatin versus
Revascularization Treatment Investigators.
N Engl J Med. 1999;341:70–76.
This article has been cited by other articles:
 |
|
 |
|
  G. Nickenig and J.-M. Sinning Response to drug-eluting stents do we need drugs to recompense drug elution? J. Am. Coll. Cardiol., December 8, 2009; 54(24): 2330 - 2332. [Full Text] [PDF]
|
|
|
|
 |
|
 V. Prinz and M. Endres The Acute (Cerebro)Vascular Effects of Statins Anesth. Analg., August 1, 2009; 109(2): 572 - 584. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. H. Schindler, R. Campisi, D. Dorsey, J. O. Prior, M. Olschewski, J. Sayre, and H. R. Schelbert Effect of hormone replacement therapy on vasomotor function of the coronary microcirculation in post-menopausal women with medically treated cardiovascular risk factors Eur. Heart J., April 2, 2009; 30(8): 978 - 986. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Westphal, C. Abletshauser, and C. Luley Fluvastatin Treatment and Withdrawal: Effects on Endothelial Function Angiology, October 1, 2008; 59(5): 613 - 618. [Abstract] [PDF]
|
|
|
|
|
|
T. Herrler, M. Bohm, and C. Heeschen More good reasons for adherence to statin therapy during acute coronary syndromes Eur. Heart J., September 1, 2008; 29(17): 2061 - 2063. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Rodriguez-Yanez, J. Agulla, R. Rodriguez-Gonzalez, T. Sobrino, and J. Castillo Review: Statins and stroke Therapeutic Advances in Cardiovascular Disease, June 1, 2008; 2(3): 157 - 166. [Abstract] [PDF]
|
|
|
|
 |
|
 F. Gao, L. Linhartova, A. McD. Johnston, and D. R. Thickett Statins and sepsis Br. J. Anaesth., March 1, 2008; 100(3): 288 - 298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Rosengarten, D. Auch, and M. Kaps Effects of Initiation and Acute Withdrawal of Statins on the Neurovascular Coupling Mechanism in Healthy, Normocholesterolemic Humans Stroke, December 1, 2007; 38(12): 3193 - 3197. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Blanco, F. Nombela, M. Castellanos, M. Rodriguez-Yanez, M. Garcia-Gil, R. Leira, I. Lizasoain, J. Serena, J. Vivancos, M. A. Moro, et al. Statin treatment withdrawal in ischemic stroke: A controlled randomized study Neurology, August 28, 2007; 69(9): 904 - 910. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Nohria, M. E. Grunert, Y. Rikitake, K. Noma, A. Prsic, P. Ganz, J. K. Liao, and M. A. Creager Rho Kinase Inhibition Improves Endothelial Function in Human Subjects With Coronary Artery Disease Circ. Res., December 8, 2006; 99(12): 1426 - 1432. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. M. Greer, A. K. Kakkar, J. W. Elrod, L. J. Watson, S. P. Jones, and D. J. Lefer Low-dose simvastatin improves survival and ventricular function via eNOS in congestive heart failure Am J Physiol Heart Circ Physiol, December 1, 2006; 291(6): H2743 - H2751. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. H. Zucker Novel Mechanisms of Sympathetic Regulation in Chronic Heart Failure Hypertension, December 1, 2006; 48(6): 1005 - 1011. [Full Text] [PDF]
|
|
|
|
|
|
M. Endres and U. Laufs Discontinuation of Statin Treatment in Stroke Patients Stroke, October 1, 2006; 37(10): 2640 - 2643. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. P. van de Visse, M. van der Heijden, J. Verheij, G. P. van Nieuw Amerongen, V. W. M. van Hinsbergh, A. R. J. Girbes, and A. B. J. Groeneveld Effect of prior statin therapy on capillary permeability in the lungs after cardiac or vascular surgery. Eur. Respir. J., May 1, 2006; 27(5): 1026 - 1032. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. A. Kidd, H. Hong, A. Majid, D. I. Kaufman, and A. F. Chen Inhibition of Brain GTP Cyclohydrolase I and Tetrahydrobiopterin Attenuates Cerebral Infarction via Reducing Inducible NO Synthase and Peroxynitrite in Ischemic Stroke Stroke, December 1, 2005; 36(12): 2705 - 2711. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Madonna, P. Di Napoli, M. Massaro, A. Grilli, M. Felaco, A. De Caterina, D. Tang, R. De Caterina, and Y.-J. Geng Simvastatin Attenuates Expression of Cytokine-inducible Nitric-oxide Synthase in Embryonic Cardiac Myoblasts J. Biol. Chem., April 8, 2005; 280(14): 13503 - 13511. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. B. Singhal, M. A. Topcuoglu, D. J. Dorer, C. S. Ogilvy, B. S. Carter, and W. J. Koroshetz SSRI and statin use increases the risk for vasospasm after subarachnoid hemorrhage Neurology, March 22, 2005; 64(6): 1008 - 1013. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Narumiya, S. Sasaki, N. Kuwahara, H. Irie, T. Kusaba, H. Kameyama, K. Tamagaki, T. Hatta, K. Takeda, and H. Matsubara HMG-CoA reductase inhibitors up-regulate anti-aging klotho mRNA via RhoA inactivation in IMCD3 cells Cardiovasc Res, November 1, 2004; 64(2): 331 - 336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Endres and U. Laufs Effects of Statins on Endothelium and Signaling Mechanisms Stroke, November 1, 2004; 35(11_suppl_1): 2708 - 2711. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. A. Spencer, G. C. Fonarow, P. D. Frederick, R. S. Wright, N. Every, R. J. Goldberg, J. M. Gore, W. Dong, R. C. Becker, W. French, et al. Early Withdrawal of Statin Therapy in Patients With Non-ST-Segment Elevation Myocardial Infarction: National Registry of Myocardial Infarction Arch Intern Med, October 25, 2004; 164(19): 2162 - 2168. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. A. Spencer, J. Allegrone, R. J. Goldberg, J. M. Gore, K. A.A. Fox, C. B. Granger, R. H. Mehta, D. Brieger, and the GRACE Investigators* Association of Statin Therapy with Outcomes of Acute Coronary Syndromes: The GRACE Study Ann Intern Med, June 1, 2004; 140(11): 857 - 866. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. E. Porter, N. A. Turner, D. J. O'Regan, A. J. Balmforth, and S. G. Ball Simvastatin reduces human atrial myofibroblast proliferation independently of cholesterol lowering via inhibition of RhoA Cardiovasc Res, March 1, 2004; 61(4): 745 - 755. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Catron, Y. Lange, J. Borensztajn, M. D. Sylvester, B. D. Jones, and K. Haldar Salmonella enterica Serovar Typhimurium Requires Nonsterol Precursors of the Cholesterol Biosynthetic Pathway for Intracellular Proliferation Infect. Immun., February 1, 2004; 72(2): 1036 - 1042. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Varela and J. L. Garvin Acute and Chronic Regulation of Thick Ascending Limb Endothelial Nitric Oxide Synthase by Statins J. Am. Soc. Nephrol., February 1, 2004; 15(2): 269 - 275. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Werba, E. Tremoli, P. Massironi, M. Camera, A. Cannata, F. Alamanni, P. Biglioli, and A. Parolari Statins in coronary bypass surgery: rationale and clinical use Ann. Thorac. Surg., December 1, 2003; 76(6): 2132 - 2140. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R.P. Brandes, S. Beer, T. Ha, and R. Busse Withdrawal of Cerivastatin Induces Monocyte Chemoattractant Protein 1 and Tissue Factor Expression in Cultured Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, October 1, 2003; 23(10): 1794 - 1800. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Almog Statins, Inflammation, and Sepsis: Hypothesis Chest, August 1, 2003; 124(2): 740 - 743. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Dechend, J. Gieffers, R. Dietz, A. Joerres, J. Rupp, F. C. Luft, and M. Maass Hydroxymethylglutaryl Coenzyme A Reductase Inhibition Reduces Chlamydia pneumoniae-Induced Cell Interaction and Activation Circulation, July 22, 2003; 108(3): 261 - 265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Davis, H. Cai, L. McCann, T. Fukai, and D. G. Harrison Role of c-Src in regulation of endothelial nitric oxide synthase expression during exercise training Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1449 - H1453. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Houten, M. S. Schneiders, R. J. A. Wanders, and H. R. Waterham Regulation of Isoprenoid/Cholesterol Biosynthesis in Cells from Mevalonate Kinase-deficient Patients J. Biol. Chem., February 14, 2003; 278(8): 5736 - 5743. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Gertz, U. Laufs, U. Lindauer, G. Nickenig, M. Bohm, U. Dirnagl, and M. Endres Withdrawal of Statin Treatment Abrogates Stroke Protection in Mice Stroke, February 1, 2003; 34(2): 551 - 557. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
The ENCORE Investigators* Effect of Nifedipine and Cerivastatin on Coronary Endothelial Function in Patients With Coronary Artery Disease: The ENCORE I Study (Evaluation of Nifedipine and Cerivastatin On Recovery of coronary Endothelial function) Circulation, January 28, 2003; 107(3): 422 - 428. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Stamatakis, E. Cernuda-Morollon, O. Hernandez-Perera, and D. Perez-Sala Isoprenylation of RhoB Is Necessary for Its Degradation. A NOVEL DETERMINANT IN THE COMPLEX REGULATION OF RhoB EXPRESSION BY THE MEVALONATE PATHWAY J. Biol. Chem., December 13, 2002; 277(51): 49389 - 49396. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. R. Mattingly, R. A. Gibbs, R. E. Menard, and J. J. Reiners Jr. Potent Suppression of Proliferation of A10 Vascular Smooth Muscle Cells by Combined Treatment with Lovastatin and 3-Allylfarnesol, an Inhibitor of Protein Farnesyltransferase J. Pharmacol. Exp. Ther., October 1, 2002; 303(1): 74 - 81. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Sposito and M. J. Chapman Statin Therapy in Acute Coronary Syndromes: Mechanistic Insight Into Clinical Benefit Arterioscler Thromb Vasc Biol, October 1, 2002; 22(10): 1524 - 1534. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Hippenstiel, B. Schmeck, P. D. N'Guessan, J. Seybold, M. Krull, K. Preissner, C. V. Eichel-Streiber, and N. Suttorp Rho protein inactivation induced apoptosis of cultured human endothelial cells Am J Physiol Lung Cell Mol Physiol, October 1, 2002; 283(4): L830 - L838. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. D. Waters, G. G. Schwartz, A. G. Olsson, A. Zeiher, M. F. Oliver, P. Ganz, M. Ezekowitz, B. R. Chaitman, S. J. Leslie, T. Stern, et al. Effects of Atorvastatin on Stroke in Patients With Unstable Angina or Non-Q-Wave Myocardial Infarction: A Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering (MIRACL) Substudy Circulation, September 24, 2002; 106(13): 1690 - 1695. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Schomig, J. Mehilli, H. Holle, K. Hosl, D. Kastrati, J.u. Pache, M. Seyfarth, F.-J. Neumann, J. Dirschinger, and A. Kastrati Statin treatment following coronary artery stenting and one-year survival J. Am. Coll. Cardiol., September 4, 2002; 40(5): 854 - 861. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Bates, C. E. Ruggeroli, S. Goldman, and M. A. Gaballa Simvastatin restores endothelial NO-mediated vasorelaxation in large arteries after myocardial infarction Am J Physiol Heart Circ Physiol, August 1, 2002; 283(2): H768 - H775. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Vecchione and R. P. Brandes Withdrawal of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors Elicits Oxidative Stress and Induces Endothelial Dysfunction in Mice Circ. Res., July 26, 2002; 91(2): 173 - 179. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. W. van Etten, E. J.P. de Koning, M. L. Honing, E. S. Stroes, C. A. Gaillard, and T. J. Rabelink Intensive Lipid Lowering by Statin Therapy Does Not Improve Vasoreactivity in Patients With Type 2 Diabetes Arterioscler Thromb Vasc Biol, May 1, 2002; 22(5): 799 - 804. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Chu, D. D. Heistad, K. L. Knudtson, K. G. Lamping, and F. M. Faraci Quantification of mRNA for Endothelial NO Synthase in Mouse Blood Vessels by Real-Time Polymerase Chain Reaction Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 611 - 616. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ledoux, D. Laouari, M. Essig, I. Runembert, G. Trugnan, J.B. Michel, and G. Friedlander Lovastatin Enhances Ecto-5'-Nucleotidase Activity and Cell Surface Expression in Endothelial Cells: Implication of Rho-Family GTPases Circ. Res., March 8, 2002; 90(4): 420 - 427. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Dechend, D. Muller, J. K. Park, A. Fiebeler, H. Haller, and F. C. Luft Statins and angiotensin II-induced vascular injury Nephrol. Dial. Transplant., March 1, 2002; 17(3): 349 - 353. [Full Text] [PDF]
|
|
|
|
|
|
U. Laufs, H. Kilter, C. Konkol, S. Wassmann, M. Bohm, and G. Nickenig Impact of HMG CoA reductase inhibition on small GTPases in the heart Cardiovasc Res, March 1, 2002; 53(4): 911 - 920. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Santizo, H.-L. Xu, E. Galea, S. Muyskens, V. L. Baughman, and D. A. Pelligrino Combined Endothelial Nitric Oxide Synthase Upregulation and Caveolin-1 Downregulation Decrease Leukocyte Adhesion in Pial Venules of Ovariectomized Female Rats Stroke, February 1, 2002; 33(2): 613 - 616. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. L. Kanabrocki, M. George, R. C. Hermida, H. L. Messmore, M. D. Ryan, D. E. Ayala, D. A. Hoppensteadt, J. Fareed, F. W. Bremner, J. L. H. C. Third, et al. Day-Night Variations in Blood Levels of Nitric Oxide, T-TFPI, and E-Selectin Clinical and Applied Thrombosis/Hemostasis, October 1, 2001; 7(4): 339 - 345. [Abstract] [PDF]
|
|
|
|
 |
|
 S. Beqaj, S. Jakkaraju, R. R. Mattingly, D. Pan, and L. Schuger High RhoA activity maintains the undifferentiated mesenchymal cell phenotype, whereas RhoA down-regulation by laminin-2 induces smooth muscle myogenesis J. Cell Biol., March 4, 2002; 156(5): 893 - 903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ledoux, D. Laouari, M. Essig, I. Runembert, G. Trugnan, J.B. Michel, and G. Friedlander Lovastatin Enhances Ecto-5'-Nucleotidase Activity and Cell Surface Expression in Endothelial Cells: Implication of Rho-Family GTPases Circ. Res., March 8, 2002; 90(4): 420 - 427. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Chu, D. D. Heistad, K. L. Knudtson, K. G. Lamping, and F. M. Faraci Quantification of mRNA for Endothelial NO Synthase in Mouse Blood Vessels by Real-Time Polymerase Chain Reaction Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 611 - 616. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Heeschen, C. W. Hamm, U. Laufs, S. Snapinn, M. Bohm, and H. D. White Withdrawal of Statins Increases Event Rates in Patients With Acute Coronary Syndromes Circulation, March 26, 2002; 105(12): 1446 - 1452. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||