3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase and Isoprenylation Inhibitors Induce Apoptosis of Vascular Smooth Muscle Cells in Culture

From the Instituto de Investigación Médica (C.G., L.M.B.-C., M.O., A.O., J.J.P., J.E.), Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Madrid, Spain; Centro de Investigación en Sanidad Animal (C.A.), INIA, Valdeolmos, Madrid, Spain; and Parke Davis Spain (C.D., G.H.), Barcelona, Spain. The current address for Dr Guijarro is the Department of Internal Medicine, Fundación Hospital Alcorcón, Alcorcón, Madrid, Spain.

Correspondence to Jesús Egido, MD, PhD, Research Laboratories, Fundación Jiménez Díaz, Avda Reyes Católicos 2, 28040 Madrid, Spain. E-mail jegido{at}uni.fjd.es

	Abstract

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Abstract—Recent evidence suggests that apoptosis may be involved in the control of vascular smooth muscle cell (VSMC) number in atherosclerotic lesions. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been reported to induce apoptosis in a variety of tumor cell lines. To evaluate whether these agents also induce apoptosis of VSMCs, cultured rat VSMCs were treated with increasing doses of atorvastatin in the presence of FBS as a survival factor. The presence of apoptosis was evaluated by morphological criteria, annexin V binding, and DNA fragmentation and quantified as the proportion of hypodiploid cells by flow cytometry. Atorvastatin induced apoptosis in a dose-dependent manner, an effect also seen with simvastatin and lovastatin, but not with the hydrophilic drug pravastatin. The proapoptotic effect of statins was seen only when the inhibition of acetate incorporation into sterols was >95% and was fully reversed by mevalonate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate but not by isopentenyl adenosine, ubiquinone, or squalene, suggesting a role for prenylated proteins in the regulation of VSMC apoptosis. To further assess the role of protein prenylation, VSMCs were exposed to the prenyl transferase inhibitors perillic acid and manumycin A. Both agents induced VSMC apoptosis as evaluated by the above-mentioned criteria. Finally, VSMC treatment with lipophilic statins was associated with decreased prenylation of p21-Rho B, further supporting the role of protein prenylation inhibition in statin-induced VSMC apoptosis. The present data suggest that interference with protein prenylation by HMG-CoA reductase inhibitors or other agents may provide new strategies for the prevention of neointimal thickening.

Key Words: smooth muscle cell • apoptosis • mevalonate • protein isoprenylation • Rho • atherosclerosis

	Introduction

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Apoptosis has recently been reported to be present in human atherosclerosis and in experimental models of vascular damage and virtually absent in normal vessels, suggesting a potential role for apoptosis in the pathophysiology of vascular injury.^{1 2 3 4 5 6} Human vascular smooth muscle cells (VSMCs) from atheromatous lesions are more likely to undergo apoptosis than are VSMCs derived from normal arteries.⁷ In addition, apoptosis is more frequent in proliferative lesions, particularly restenosis lesions.^{3 5} The temporal sequence of proliferation and apoptosis in experimental models is consistent with a role for apoptosis in the control of neointimal cellularity.¹ Accordingly, VSMC apoptosis has been proposed to play a significant role in the control of neointimal thickening.^{5 6 8} In spite of its potential importance, little is known regarding the mechanisms controlling VSMC apoptosis in vivo.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins, Figure 1) have been shown to be efficacious in reducing cardiovascular morbidity and mortality in primary and secondary prevention clinical trials.^{9 10 11} Although the salutary effects of these agents may be explained by their beneficial actions on the lipid profile, increasing evidence suggests that statins may also exhibit effects unrelated to lipid reduction (Figure 1).^{12 13 14} In experimental models, HMG-CoA reductase inhibitors reduced neointimal thickening regardless of the effect of cholesterol.^{15 16 17 18} Neointimal VSMC number is the result of the migration of VSMCs from the media and their subsequent proliferation and eventual death, including programmed cell death.^{19 20} Much of the attention on the effects of statins on VSMC biology has been devoted to migration and proliferation.^{16 21 22 23 24 25} Indeed, both phenomena can be attenuated by HMG-CoA reductase inhibition and have been proposed as potential mechanisms of action in the prevention of vascular damage by statins. HMG-CoA reductase inhibitors induce programmed cell death in a variety of cell lines.^{26 27 28 29 30} In spite of its potential relevance, no attempts to evaluate the effect of HMG-CoA reductase inhibitors on VSMC apoptosis have been reported. In the present study, we show that lipophilic HMG-CoA reductase inhibitors induce apoptosis of VSMCs even in the presence of survival factors. This effect is paralleled by an attenuation of the prenylation of p21-Rho B and is reversed by the addition of mevalonate, farnesyl pyrophosphate (F-PP), and/or geranylgeranyl pyrophosphate (GG-PP), suggesting a role for isoprenylated proteins such as Rho B in the apoptosis of VSMCs induced by statins. Our data suggest that interference with protein prenylation by HMG-CoA reductase inhibitors or other agents may provide new strategies for the prevention of neointimal thickening.

View larger version (12K): [in this window] [in a new window]   Figure 1. The mevalonate pathway. Schematic representation of the mevalonate pathway, depicting the main enzymes (in italics), mevalonate metabolites, and the site of action of HMG-CoA reductase inhibitors. PP indicates pyrophosphate; tGeranylgeranyl-PP, all trans geranylgeranyl-PP; and cGeranylgeranyl-PP, cis-trans-trans geranylgeranyl-PP.

	Materials and Methods

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Reagents and Chemicals
DMEM, penicillin, streptomycin, and trypsin-EDTA were from Bio Whitaker. FBS was from GIBCO. Atorvastatin (sodium salt, dissolved in Tris borate, pH 8) was obtained from Parke Davis. Lovastatin and simvastatin (from Merck Sharp and Dohme Spain) were converted to the active compounds as described by Kita et al.³¹ Pravastatin was obtained from Bristol Myers-Squibb Spain and dissolved in NaCl (155 mmol/L). Perillic acid and manumycin A (from Sigma Chemical Co) were dissolved in ethanol and DMSO, respectively. FITC annexin V was kindly provided by Dr J. Turnay (School of Chemistry, Universidad Complutense, Madrid, Spain). [³H]Acetic acid (sodium salt; specific activity, 2.52 Ci/mmol) was from DuPont NEN. Rabbit anti–Rho B polyclonal antibody (Sc-180) was from Santa Cruz Biotechnology. The remaining reagents were obtained from Sigma unless specified otherwise.

VSMC Culture
Rat thoracic aorta smooth muscle cells were isolated essentially as described by Owens et al.³² Briefly, male Sprague-Dawley rats weighing 200 to 250 g were killed by an overdose of pentobarbital, and the thoracic aorta was aseptically excised and placed in PBS. Adhering fat and connective tissue were removed by blunt dissection. The aorta was opened longitudinally and preincubated in DMEM containing collagenase (type II, 290 U/mL), penicillin (100 U/mL), and streptomycin (100 µg/mL) for 15 to 20 minutes at 37°C in 95% air/5% CO₂. After dissection, aortas were placed in fresh enzyme solution, minced into 1-mm pieces, and incubated for an additional 1.5 to 2 hours. Pieces were rinsed twice with PBS to remove the cells, and PBS was then centrifuged. The cells were resuspended in DMEM with antibiotics and 10% FBS, counted, and seeded at a concentration of 10⁴ cells/cm² in plastic culture flasks (Costar). Cells were characterized as smooth muscle cells by their typical hill-and-valley morphology by phase-contrast microscopy and by positive immunostaining for -smooth muscle actin (clone 1A4, Sigma). Media were replaced every 2 to 3 days. At confluence, cells were harvested for passaging with trypsin-EDTA. Cells between passages 3 and 12 were used for all the experiments.

Cell DNA Staining
Adherent cells were washed once with PBS and incubated with PBS containing 9.4 µmol/L bisbenzimide (Hoechst 33342) for 20 minutes in the dark and examined under fluorescence microscopy. Floating cells were collected by centrifugation, and a cell smear was stained with bisbenzimide as indicated above.

Flow Cytometry Assessment of Cell Death
Cellular DNA content was assessed by flow cytometry. For this purpose, cells were cultured in 12-well plates and treated as appropriate. Cells attached to the plate were collected with trypsin and 2.2 mmol/L EDTA in PBS and mixed with detached cells present in the supernatant. Cells were spun and resuspended in a solution containing 75 µmol/L propidium iodide, 10 mg/L RNase A, and 0.05% Nonidet P-40 in PBS; then they were incubated at 4°C for 30 minutes in the dark and analyzed by flow cytometry (Coulter EPICS XL-MCL flow cytometer, Hialeah, FL) using LYSIS II software.^{33 34} The percentage of cells with decreased DNA staining (A₀), composed of apoptotic cells resulting from either fragmentation or decreased chromatin, of a minimum of 5000 to 10 000 cells per experimental condition was counted. Cell debris were excluded from analysis by selective gating based on anterior and right angle scatter. As assessed by flow cytometry, none of the solvents of the different compounds, up to the highest dose used in our experimental conditions, induced any significant degree of apoptosis.

DNA Electrophoresis
For the evaluation of DNA fragmentation, 10 million cells were incubated under the different experimental conditions for 48 hours and assayed essentially as described.³⁵ At the end of this period, floating and adherent cells were collected, rinsed twice with cold PBS, and lysed by incubation in 20 mmol/L EDTA, 50 mmol/L Tris-HCl (pH 8), and 0.5% (wt/vol) sodium lauroylsarcosine for 30 minutes at 4°C. Cell debris and high-molecular-weight DNA were precipitated by centrifugation at 12 000g for 30 minutes at 4°C. Supernatants containing fragmented DNA were then treated with proteinase K (0.2 g/L) at 50°C for 1 hour and 2 g/L RNase A at 50°C for another hour and run in a 1.5% agarose gel containing ethidium bromide. Gels were examined and photographed under UV light.

Flow Cytometry Assessment of Annexin V Binding
Annexin V binding was studied essentially as described by Vermes et al,³⁶ with minor modifications. For that purpose, cells were cultured in 12-well plates and treated as appropriate. Cells attached to the plate were carefully collected with 2.2 mmol/L EDTA in PBS and mixed with detached cells present in the supernatant. Cells were spun, rinsed with PBS, and resuspended in a solution containing 1.5 mg/L FITC annexin V, 10 mmol/L HEPES, 150 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl₂, and 1.8 mmol/L CaCl₂ for 15 minutes at 37°C. Cells were washed twice with the same buffer (without FITC annexin V) and analyzed by flow cytometry (Coulter EPICS XL-MCL flow cytometer). Green fluorescence intensity of at least 2000 to 5000 events is displayed on a logarithmic scale against cell number. Cell debris were excluded from analysis.

[³H]Acetate Incorporation Into Sterols
Approximately 5 million subconfluent exponentially growing smooth muscle cells were incubated with different concentrations of HMG-CoA reductase inhibitors in DMEM containing 10% FBS for 3 hours. At the end of this time, 5 µCi of [³H]sodium acetate was added, and cells were incubated for an additional period of 21 hours. Afterward, cells were collected and rinsed twice with PBS, and lipids were extracted by the method of Bligh and Dyer.³⁷ The chloroform fractions were dried under N₂, resuspended in 2 mL of 1 mol/L KOH in 95% ethanol, and saponified at 90°C for 50 minutes. Nonsaponifiable lipids were extracted twice with 1.2 mL of hexane and dried under N₂. Four hundred micrograms of cholesterol and 2 mL of isopropanol were added to each sample. Sterols were then precipitated by the addition of 1 mL of digitonin reagent (1% digitonin in 50% ethanol) and incubation at 4°C for 30 minutes.³⁸ Samples were spun, and the precipitate was washed twice with cold acetone and resuspended in 2 mL of methanol. Precipitates were extensively resuspended with 2 additional washes with methanol, pooled, and dried before counting in scintillation vials.

Immunoblotting
Cells from different experimental conditions were collected, rinsed twice with cold PBS, and pelleted. Cells were then briefly sonicated in 1 mL of PBS containing 2 mmol/L EDTA, 2 mmol/L phenylmethylsulfonyl fluoride, and 1 µmol/L pepstatin A. Cell membranes were collected by centrifugation at 50 000g for 30 minutes and resuspended in 100 mmol/L Tris-HCl, 300 mmol/L NaCl, 1% Triton X-100, and 0.1% SDS containing 2 mmol/L EDTA, 2 mmol/L phenylmethylsulfonyl fluoride, and 1 µmol/L pepstatin A. Protein in membrane or soluble extracts was calculated by the BCA method (Pierce). Equal amounts of protein (50 to 100 µg) were loaded into 12% acrylamide gels and electrophoresed as described.³⁹ The resolved proteins were transferred onto PVDF membranes (Immobilon, Millipore). The nonspecific sites of the membrane were blocked by incubation at room temperature 1 hour in 7.5% nonfat dry milk powder in PBS containing 0.1% Tween 20 (PBST). The membrane was incubated overnight at 4°C with rabbit anti–Rho B polyclonal antibody (Sc-180, Santa Cruz Biotechnology) in PBST containing 5% nonfat dry milk. The membrane was washed with PBST and incubated 1 hour at room temperature with horseradish peroxidase–conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology) in PBST containing 5% nonfat dry milk. The membrane was then washed with PBST and incubated 30 minutes in PBST containing 400 mmol/L NaCl, followed by detection with enhanced chemiluminescence (ECL kit, Amersham). Films were scanned on a densitometer and quantified using ImageQant software (Molecular Dynamics).

Data Presentation
Representative data from 2 to 5 independent experiments are presented. For quantitative data, the mean±SD of triplicate or quadruplicate samples from 1 of 3 to 5 independent experiments is presented. For the comparison of group means, 1-way ANOVA and Student-Newman-Keuls tests were used as appropriate. A value P<0.05 was considered statistically significant.

	Results

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Atorvastatin and Lipophilic HMG-CoA Reductase Inhibitors Induce Apoptosis of VSMCs
To evaluate whether HMG-CoA reductase inhibitors induce apoptosis of VSMCs, exponentially growing subconfluent rat VSMCs were exposed to increasing concentrations of the HMG-CoA reductase inhibitor atorvastatin for 24 hours in the presence of 10% FBS as a survival factor. Atorvastatin treatment was associated with cell rounding and detachment from the plate substrate and surrounding cells, as assessed by inverted phase microscopy, in a dose-dependent manner (Figure 2). Staining with DNA-binding dyes revealed that treatment of VSMCs with atorvastatin was associated with the appearance of chromatin condensation and fragmentation characteristic of apoptosis (Figure 2). These changes were particularly prominent in detached cells floating in the culture media. To further confirm that atorvastatin induced apoptosis of VSMCs, the cell DNA content was assessed by flow cytometry. Atorvastatin treatment was associated with the appearance of a cell population with a reduced DNA content (hypodiploid), usually referred to as sub G₁ or A₀, characteristic of apoptosis (Figures 3 to 5). Most dramatic reductions of DNA content were evident in floating cells (not shown), mirroring the effects on chromatin fragmentation. The reduction in cellular DNA content was paralleled by the appearance of DNA fragmentation into multiples of 180 to 200 kb, constituting a typical laddering pattern characteristic of apoptosis (Figure 6). Finally, atorvastatin treatment was also associated with increased binding of annexin V, a recently described early indicator of apoptosis (Figure 7).³⁶ Again, most of the increased annexin V binding was attributable to floating cells. Mevalonate, in a dose-dependent manner, completely reversed the effects of atorvastatin in cell morphology, DNA content, and fragmentation, and annexin V binding (Figures 2 to 4, 6, and 7), suggesting that the effects of atorvastatin were indeed related to the inhibition of the mevalonate pathway. To ascertain whether the effects of atorvastatin were shared by other HMG-CoA reductase inhibitors, VSMCs were incubated with increasing concentrations of 2 lipophilic (simvastatin and lovastatin) and 1 hydrophilic (pravastatin) HMG-CoA reductase inhibitor. Both simvastatin and lovastatin produced changes similar to those observed with atorvastatin with an even higher potency, whereas pravastatin (up to 100 µmol/L) failed to induce any of the above-mentioned effects (Figures 4 and 6). In every case, the addition of mevalonate completely reversed the statin-induced effects (Figures 4 and 6).

View larger version (74K): [in this window] [in a new window]   Figure 2. Morphological effects of atorvastatin and isoprenylation inhibitors in VSMCs in culture. Exponentially growing rat VSMCs were exposed for 24 hours to atorvastatin (100 µmol/L), atorvastatin+mevalonate (100 µmol/L), perillic acid (10 mmol/L, in the presence of atorvastatin+mevalonate), or manumycin (50 µmol/L), as indicated. A, Phase-contrast microscopic photographs (original magnification, x150). B and C, Fluorescence photomicrographs of attached (B) and detached (C) cells after DNA staining with 9.4 µmol/L bisbenzimide (Hoechst 33342). Representative examples of at least 5 independent experiments are shown. Treatment with atorvastatin, perillic acid, and manumycin was associated different degrees of cell rounding and detachment from substrate and surrounding cells. DNA staining revealed that these changes were associated with increased chromatin condensation and fragmentation (B), characteristic of apoptosis, that were particularly prominent in floating cells (C). All the atorvastatin-induced effects were reversed by the addition of mevalonate.

View larger version (23K): [in this window] [in a new window]   Figure 3. Dose-response effects of atorvastatin and mevalonate. Exponentially growing rat VSMCs were exposed to increasing concentrations of atorvastatin (open bars [panel B]) for 24 hours. Some cells were also treated with increasing concentrations of mevalonate in addition to 100 µmol/L atorvastatin (ATV+mevalonate, solid bars [panel B]). Adherent cells were collected by trypsinization, mixed with detached cells, and subjected to flow cytometric analysis, as indicated in Materials and Methods. A, Representative flow cytometric analysis of cell cycle in control cells and cells treated with 100 µmol/L atorvastatin±100 µmol/L mevalonate, as indicated. B, Histograms representing the mean±SD proportion of cells with hypodiploid DNA content (A0) of triplicate samples. Atorvastatin treatment was associated, in a dose-dependent manner, with an increased proportion of cells with hypodiploid DNA content, detected in cell cycle analysis (A0, arrow [panel A]), characteristic of apoptotic cells. The effects of atorvastatin were fully reversed by mevalonate.

View larger version (34K): [in this window] [in a new window]   Figure 4. Effects of different HMG-CoA reductase inhibitors on apoptosis and sterol synthesis. Top, Exponentially growing rat VSMCs (control, open bar) were exposed to increasing concentrations of several HMG-CoA reductase inhibitors (statins) in the absence (hatched bars) or presence (solid bars) of mevalonate (100 µmol/L). Cells were collected and analyzed as in Figure 3. Histograms represent the mean±SD proportion of cells with hypodiploid DNA content (A0) of triplicate samples. Values are representative of 3 independent experiments. *P<0.05 vs control. Bottom, Exponentially growing rat VSMCs were treated with different concentrations of HMG-CoA reductase inhibitors, and the 21-hour incorporation of [3H]acetate into sterols was measured, as described in Materials and Methods (control=100%). Values are representative of 2 independent experiments. *P<0.05 vs control. Atorvastatin, lovastatin, and simvastatin treatment induced, in a dose-dependent manner, the appearance of an increased proportion of cells with hypoploid DNA content (A0, arrow), characteristic of apoptosis. These effects were fully reversed by mevalonate. In contrast, pravastatin treatment did not increase the proportion of cells with reduced DNA content. Changes consistent with apoptosis were observed only when sterol synthesis was inhibited by >95%.

View larger version (31K): [in this window] [in a new window]   Figure 5. F-PP and GG-PP reverse the effects of atorvastatin. Exponentially growing rat VSMCs were exposed to 100 µmol/L atorvastatin for 24 hours in the absence (solid bars [panel B]) or presence of different concentrations of F-PP (dotted bars [panel B]) or GG-PP (hatched bars [panel B]). Cell collection and data expression are as in Figure 3. A, Representative flow cytometric analysis of cell cycle of control cells and cells exposed to 100 µmol/L atorvastatin in the presence or absence of 5 µmol/L F-PP or 4 µmol/L GG-PP, as indicated. B, Histograms representing the mean±SD proportion of cells with hypodiploid DNA content (A0) of triplicate samples. ATV+F-PP and ATV+GG-PP indicate atorvastatin plus F-PP and GG-PP, respectively. Values are representative of 4 independent experiments. Both F-PP and GG-PP reversed, in a dose-dependent manner, the effects of atorvastatin on the proportion of cells with reduced DNA content (A0, arrow [panel A]), a characteristic feature of apoptosis.

View larger version (58K): [in this window] [in a new window]   Figure 6. Effects of HMG-CoA reductase and isoprenylation inhibitors on DNA fragmentation. Reversal by isoprenoids. Exponentially growing rat VSMCs were exposed to atorvastatin (0 to 100 µmol/L), other statins (100 µmol/l), perillic acid (10 mmol/L, in the presence of 100 µmol/L atorvastatin+100 µmol/L mevalonate [MVA]), or manumycin A (50 µmol/L), as indicated. Some cells were exposed to 100 µmol/L MVA, 10 µmol/L F-PP, or 10 µmol/L GG-PP in addition to 100 µmol/L atorvastatin, as indicated. Fragmented DNA was extracted, run in a 1.5% agarose gel, stained with ethidium bromide, and photographed under UV light as described in Materials and Methods. As shown, treatment with atorvastatin was associated, in a dose-dependent manner, with the appearance of a 180- to 200-bp DNA fragmentation pattern, typical of apoptosis. This effect of atorvastatin was virtually abolished by either MVA, F-PP, or GG-PP. Similarly, treatment with lipophilic statins (but not pravastatin) or isoprenylation inhibitors was also associated with the appearance of a DNA laddering pattern.

View larger version (28K): [in this window] [in a new window]   Figure 7. Effects of HMG-CoA reductase and isoprenylation inhibitors on annexin V binding. Exponentially growing rat VSMCs were exposed to 30 µmol/L lovastatin, 100 µmol/L atorvastatin±100 µmol/L mevalonate, perillic acid 10 mmol/L (in the presence of 100 µmol/L atorvastatin+100 µmol/L mevalonate), or 50 µmol/L manumycin A (alone) for 24 hours, as indicated. Floating cells were collected, mixed with adherent cells, exposed to FITC annexin V, and analyzed by fluorescence flow cytometry, as described in Materials and Methods. Floating and adherent atorvastatin-treated cells were also analyzed separately. Numbers in different panels represent the median fluorescence intensity per experimental condition. Values are representative of 2 independent experiments. Note that VSMC treatment with all the tested drugs was associated with increased FITC–annexin V binding, as assessed by a right shift in the curve of fluorescence intensity. Annexin V binds phosphatidylserine on the outer layer of cell membrane and, as such, is considered an indicator of the loss of cell membrane polarity, an early feature of apoptosis. Not surprisingly, most of the increased annexin V binding was attributable to floating cells, as depicted for atorvastatin-treated cells. Again, mevalonate reversed the atorvastatin-induced effects. Interestingly, the intensity of the shift of the curves parallels the previous findings on the effect of the drugs on the proportion of hypodiploid cells, ie, lovastatin>atorvastatin and manumycin A>perillic acid.

We further assessed whether the above-described effects were indeed related to the degree of inhibition of the mevalonate pathway. First, the antiapoptotic effect of mevalonate was specific for statins, since mevalonate failed to prevent VSMC apoptosis induced by other agents, such as pyrrolidinedithiocarbamate or manumycin A (not shown). Second, the proapoptotic effect of statins was very closely related to the degree of inhibition of the mevalonate pathway, as assessed by the inhibition of the incorporation of [³H]acetate into sterols. Indeed, apoptosis was present only when the inhibition of sterol synthesis was >95% (Figure 4, bottom). All the above data strongly suggest that the depletion of some mevalonate metabolite(s) is involved in the statin-induced apoptosis of VSMCs.

Mevalonate Metabolites and Apoptosis of VSMCs
To evaluate which among mevalonate metabolites may be important in atorvastatin-induced apoptosis, the reversal of atorvastatin effects by several mevalonate derivatives (Figure 1) was assessed next. Neither cholesterol (present in the serum-supplemented media) nor its precursor, squalene (1 to 25 µmol/L), prevented atorvastatin-induced apoptosis (not shown), suggesting that sterols do not play a prominent role in our experimental conditions. Similarly, neither isopentenyl adenosine (1 to 25 µmol/L) nor ubiquinone (coenzyme Q10, 1 to 25 µmol/L) reversed the atorvastatin-induced apoptosis of VSMCs (not shown). In contrast, both F-PP and GG-PP reversed, in a dose-dependent manner, the atorvastatin effects in cell morphology, DNA content and fragmentation, and annexin V binding (Figures 5 to 7).

Isoprenylation Inhibition and Apoptosis of VSMCs
Since both F-PP and GG-PP are used for the posttranslational modification of several cell proteins, we next evaluated the effects of 2 inhibitors of protein prenylation: perillic acid and manumycin A. In atorvastatin-treated mevalonate-repleted cells, the weak inhibitor of prenylation perillic acid induced, in a dose dependent manner, the appearance of the morphological changes, annexin V binding, DNA content reduction, and laddering characteristic of apoptosis in a significant, albeit small, proportion of VSMCs (Figures 2, 7, and 8). In agreement with its higher inhibitory potency, manumycin A induced, in a dose-dependent manner, apoptosis in a higher proportion of VSMCs (Figures 2, 7, and 8). As expected, the addition of exogenous mevalonate (100 µmol/L) did not modify the manumycin-induced effects (not shown). Another farnesyl transferase inhibitor, -hydroxyfarnesyl phosphonic acid, also induced in a dose-dependent manner the appearance of VSMC apoptosis (not shown).

View larger version (30K): [in this window] [in a new window]   Figure 8. Dose-response effects of protein prenylation inhibitors. Exponentially growing rat VSMCs (control, open bar [panel B]) were exposed to atorvastatin (ATV, hatched bar [panel B]), perillic acid (2 to 10 mmol/L in the presence of 100 µmol/L atorvastatin+100 µmol/L mevalonate, crosshatched bars [panel B]), or manumycin A alone (12.5 to 100 µmol/L, solid bars [panel C]) for 24 hours. A, Representative flow cytometric analysis of cell cycle in control cells and cells treated with 10 mmol/L perillic acid or 50 µmol/L manumycin, as indicated. B and C, Histograms representing the mean±SD proportion of cells with hypodiploid DNA content (A0) of triplicate samples. Values are representative of 3 independent experiments. Perillic acid treatment (in the presence of ATV-treated mevalonate [MVA]-repleted cells) was associated in a dose-dependent manner with the appearance of a small proportion of cells with a reduced DNA content (A0, arrow [panel A]), characteristic of apoptosis. Manumycin treatment resulted in a higher proportion of cells exhibiting features consistent with apoptosis.

HMG-CoA Reductase Inhibition and Protein Prenylation
The above-presented data suggest that inhibition of protein prenylation may be important in VSMC apoptosis. Therefore, we evaluated whether HMG-CoA reductase treatment was in fact associated with changes in the level of prenylation of the protein p21-Rho B, a protein that has been found to play an important role in apoptosis. Under normal conditions p21-Rho is anchored into the cell membrane by its covalent binding to either farnesyl or geranylgeranyl. When protein prenylation is inhibited, Rho B remains in the cytoplasm. The evaluation of the degree of presence of Rho B in the membrane or cytoplasmic compartments of the cells is therefore an indirect assessment of its degree of prenylation. As shown in Figure 9, treatment of VSMCs with lipophilic statins, but not pravastatin, was associated with an attenuation of the presence of Rho B in the membrane and its appearance in the cytoplasmic fraction. Interestingly, the degree of inhibition of Rho B prenylation (as assessed by its compartmentalization) by different statins closely parallels the proapoptotic effect of statins. Again, treatment with mevalonate, F-PP, or GG-PP was associated with the disappearance of Rho from the cytoplasm and its restoration into the membrane fraction.

View larger version (22K): [in this window] [in a new window]   Figure 9. Effect of statins on the prenylation of Rho B. Exponentially growing rat VSMCs were exposed to increasing concentrations of several HMG-CoA reductase inhibitors (statins, 100 µmol/L for all of them) in the absence or presence of 100 µmol/L mevalonate (MVA), 5 µmol/L F-PP, or 5 µmol/L GG-PP, as indicated. Equal amounts of membrane-bound and cytosolic proteins from the different experimental conditions were immunoblotted for Rho B, as described in Materials and Methods. Treatment with lipophilic statins (atorvastatin, lovastatin, and simvastatin) was associated with an attenuation of Rho B prenylation, as assessed by a reduced amount of Rho B in the membrane fractions and its appearance in the cytoplasmic fractions. The presence of either MVA, F-PP, or GG-PP in the cultures essentially abrogated the statin inhibition of Rho prenylation.

	Discussion

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The present data show that HMG-CoA reductase inhibition by atorvastatin induces changes of cultured rat VSMCs characteristic of apoptosis: cell rounding, detachment from the culture substrata and adjacent cells, chromatin condensation and fragmentation, increased annexin V binding, reduction of cellular DNA content, and internucleosomal fragmentation.^{36 40} The effects are specific to the inhibition of the mevalonate pathway, since the addition of exogenous mevalonate completely reversed the atorvastatin-induced effects. Conversely, the antiapoptotic effect of mevalonate is restricted to statins: it is ineffective in reversing apoptosis induced by other agents, such as pyrrolidinedithiocarbamate or manumycin. These proapoptotic effects are not restricted to atorvastatin, as 2 other agents of the same family, namely, simvastatin and lovastatin, produced similar qualitative effects. In contrast, the hydrophilic drug pravastatin failed to induce any significant changes when used in equimolar amounts. Not surprisingly, the proapoptotic effects of statins were tightly correlated with the degree of inhibition of the mevalonate pathway (Figure 4). Our results are quite similar to those reported regarding the effects of different statins on VSMC proliferation.²⁵ It is conceivable that in both cases, the hydrophilic properties of pravastatin and the lack of a specific transporter in nonhepatic cells prevented an adequate access of the drug to the cytoplasm.⁴¹ In this regard, pravastatin, compared with atorvastatin, lovastatin, and simvastatin, was also less efficacious in reducing vascular injury in 2 experimental models of atherosclerosis.^{17 18 25} Whether the potential benefits of a direct action of lipophilic statins on the vascular wall may be overshadowed by an increased incidence of extrahepatic side effects is at present unknown.

HMG-CoA reductase inhibition results in the reduction of the cellular content of a variety of sterol and nonsterol mevalonate metabolites.^{42 43} Many of these metabolites play prominent roles in cell biology that could potentially be involved in the mechanisms of apoptosis. Our data suggest that a profound inhibition of the mevalonate pathway is required for the induction of apoptosis. Cholesterol was present in the media as a part of the FBS, at concentrations considered to be sufficient for basic cellular functions.⁴⁴ Therefore, cholesterol depletion, in agreement with previous reports in other cell lines,^{26 27 28 29 30} does not seem to account for the induction of apoptosis in our experimental conditions. In addition, squalene, the first mevalonate metabolite committed to sterol synthesis, failed to reverse the atorvastatin-induced apoptosis of VSMCs, suggesting that nonsterol metabolites, rather than steroids, may play a major role. Similarly, isopentenyl adenosine, the precursor of isopentenyl tRNA, failed to reverse the atorvastatin effects and, consequently, does not seem to play a prominent role in our experimental conditions. In contrast, both F-PP and GG-PP, at concentrations in the micromolar range, completely abolished atorvastatin-induced effects. GG-PP is the precursor of ubiquinone, an important natural antioxidant. Since redox processes appear to be involved in the pathophysiology of VSMC apoptosis,^{45 46} the inhibition of ubiquinone synthesis may be of relevance. However, the addition of ubiquinone did not reverse atorvastatin-induced VSMC apoptosis, suggesting that GG-PP is acting through a different mechanism. Indeed, GG-PP and F-PP are used for the posttranslational modification of several important cell proteins.^{42 43 47 48} The attachment of an isoprenoid residue to these proteins is necessary for their anchorage to cell membranes and full functionality. Prenylated proteins and, more specifically, small GTP-binding proteins are key elements in signal transduction from membrane receptors involved in proliferation and survival of VSMCs, such as growth factors, endothelin, angiotensin II, and thrombin.⁴⁹

Increasing attention has been directed in recent years to the development of prenyl transferase inhibitors in an attempt to inhibit tumor cell proliferation.^{48 50 51 52} Surprisingly, there are no reports dealing with the potential effects of prenyl transferase inhibition on VSMC apoptosis. To assess whether protein prenylation may play a role in the control of VSMC apoptosis, VSMCs were exposed to 2 unrelated inhibitors of protein prenyl transferases: perillic acid and manumycin A.^{53 54} Perillic acid and other limonene-related compounds are weak inhibitors of the prenylation of low-molecular-weight proteins and, for this reason, are commonly used in vitro in the presence of inhibitors of mevalonate synthesis and exogenous mevalonate repletion.⁵³ Under these conditions, perillic acid induced apoptosis in a dose-dependent manner in a modest proportion of cells in our experimental conditions. The effects of perillic acid alone were somewhat weaker (not shown). In contrast, the farnesyl transferase inhibitor manumycin A induced changes in cell morphology, annexin V binding, and DNA content characteristic of apoptosis in a higher proportion of cells. Perillic acid inhibits the farnesylation and geranylgeranylation of low-molecular-weight proteins, whereas manumycin A is a selective inhibitor of protein farnesylation. Although prenylated proteins are modified specifically by either farnesyl or geranylgeranyl in normal conditions,^{47 48} little is known regarding potential modifications by a different isoprenoid when the naturally occurring reaction is interfered pharmacologically. Protein prenylation is catalyzed by 3 different enzymes that recognize both the isoprenoid and the acceptor protein: farnesyl transferase and geranylgeranyl transferases I and II.⁴⁸ However, the specificity of these enzymes for their substrates is not absolute. Indeed, K-ras is normally farnesylated but can be alternatively modified by geranylgeranyl in the absence of farnesyl.⁵⁵ Similarly, the low-molecular-weight protein Rho B can be either geranylgeranylated or farnesylated by geranylgeranyl transferase I.^{48 56} Thus, it appears that proteins usually modified by GG-PP may be prenylated by F-PP and vice versa given the appropriate circumstances. These phenomena may underlie the capacity of either F-PP or GG-PP to reverse atorvastatin effects. Nevertheless, the fact that manumycin A induces apoptosis at concentrations of >50% inhibitory concentration (IC₅₀) for farnesyl transferase but well under the IC₅₀ for geranylgeranyl transferase I suggests that farnesylated proteins may be of particular importance.⁵⁴ More specifically, the inhibition of the prenylation of Rho may be critical. Indeed, the inhibition of Rho prenylation has very recently been described to produce apoptosis in fibroblasts,⁵⁷ and the membrane restoration of Rho can overcome this effect.⁵⁸ In addition, mutant constitutively active Rho prevents T-cell apoptosis,⁵⁹ whereas Rho inactivation is associated with increased T-cell apoptosis.⁶⁰ Therefore, we examined the effect of statins on the degree of Rho B prenylation in our experimental conditions. The effects of different statins on Rho prenylation were remarkably similar to their effects on VSMC apoptosis. In addition, the reversal of the inhibition of Rho prenylation by mevalonate or its derivatives was also associated with the abrogation of the proapoptotic effect of statins. On the whole, our results strongly suggest that the interference of the prenylation of proteins, such as Rho, by HMG-CoA reductase inhibition plays a prominent role in statin-induced VSMC apoptosis. Although Rho is an excellent marker of the effect of statins on protein prenylation and is a likely candidate to be involved in statin-induced apoptosis, our data cannot exclude that some other prenylated proteins may play a similar or even greater role.

It is difficult to ascertain how the present data may be of relevance to the in vivo situation. However, it should be noted that the concentrations of statins used in these and other experimental settings to induce apoptosis are similar to those used to inhibit cell proliferation.^{16 21 22 23 24 25 26 27 28 29 30} This is hardly surprising, as the mechanisms regulating cell proliferation are inextricably connected to the regulation of programmed cell death.^{49 61 62} In this regard, the induction of apoptosis of VSMCs might play a concurrent role with the inhibition of cell proliferation in the prevention of neointimal thickening, as proposed in experimental models.^{16 17} In addition, HMG-CoA reductase inhibitors were able to induce apoptosis of VSMCs even in the presence of the survival factor, ie, FBS. The effect of statins might be enhanced in circumstances in which a certain degree of apoptosis is already taking place, such as in atheromatous plaque formation. Even if statins may favor smooth muscle cell apoptosis in vivo, the potential clinical importance of this phenomenon is controversial. On the one hand, increased apoptosis may contribute to an attenuation of the neointimal thickening seen in early atherosclerosis.²⁰ On the other hand, enhanced apoptosis may contribute to increased plaque instability and hence favor the appearance of vascular events.¹⁹ Given all these considerations, the potential role of statin-induced VSMC apoptosis in the pathophysiology of atherosclerosis deserves further investigation.

In summary, HMG-CoA reductase inhibitors induce apoptosis of VSMCs in culture, probably through a reduction of isoprenoid concentration and the subsequent protein prenylation. A better understanding of the mechanisms involved in VSMC apoptosis and its pharmacological modulation may help to provide new strategies to modify the pathophysiology of atherosclerosis.

	Acknowledgments

 
This study was supported, in part, by grants from Fondo de Investigación Sanitaria (96/2021), Ministerio de Educación (SAF 97-055, PM 97/0085), Fundación Conchita Rábago de Jiménez Díaz, Fundación Iñigo Alvarez de Toledo, and Parke Davis Spain. Dr Alonso was supported by projects PB96-0552 and SC97-066. We are grateful to Dr J. Turnay for kindly providing FITC annexin V, to Dr M.A. Lasunción for his advice on sterol synthesis studies, and to Dr J. González-Cabrero for carefully reading the manuscript.

Received May 7, 1998; accepted June 11, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Han DK, Haudenschild CC, Hong MK, Tinkle BT, Leon MB, Liau G. Evidence for apoptosis in human atherogenesis and in a rat vascular injury model. Am J Pathol. 1995;147:267–277.[Abstract]

2. Geng YJ, Libby P. Evidence for apoptosis in advanced human atheroma: colocalization with interleukin-1 beta-converting enzyme. Am J Pathol. 1995;147:251–266.[Abstract]

3. Isner JM, Kearney M, Bortman S, Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation. 1995;91:2703–2711.[Abstract/Free Full Text]

4. Björkerud S, Björkerud B. Apoptosis is abundant in human atherosclerotic lesions, especially in inflammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability. Am J Pathol. 1996;149:367–380.[Abstract]

5. Bochaton Piallat ML, Gabbiani F, Redard M, Desmoulière A, Gabbiani G. Apoptosis participates in cellularity regulation during rat aortic intimal thickening. Am J Pathol. 1995;146:1059–1064.[Abstract]

6. Guijarro C, Tuñón J, Bustos C, Hernández-Presa MA, Ortego M, Egido J. La formación de la placa aterosclerosa: un proceso inflamatorio y fibroproliferativo. Clin Invest Arterioscler. 1997;9:3–14.

7. Bennett MR, Evan GI, Schwartz SM. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest. 1995;95:2266–2274.

8. Rembold C. Could atherosclerosis originate from defective smooth muscle cell death (apoptosis)? Perspect Biol Med. 1996;39:405–408.[Medline] [Order article via Infotrieve]

9. Scandinavian Simvastatin Survival Study Group. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet. 1994;344:1383–1389.[Medline] [Order article via Infotrieve]

10. Shepherd J, Cobbe SM, Ford I, Isles CG, Lorimer AR, Macfarlane PW, McKillop JH, Packard CJ, West of Scotland Coronary Prevention Study Group. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N Engl J Med. 1995;333:1301–1307.[Abstract/Free Full Text]

11. Sacks FM, Pfeffer MA, Moye LA, Rouleau JL, Rutherford JD, Cole TG, Brown L, Warnica JW, Arnold JMO, Wun C-C, Davis BR, Braunwald E, Cholesterol and Recurrent Events Trial Investigators. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Med. 1996;335:1001–1009.[Abstract/Free Full Text]

12. 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]

13. Vaughan CJ, Murphy MB, Buckley BM. Statins do more than just lower cholesterol. Lancet. 1996;348:1079–1082.[Medline] [Order article via Infotrieve]

14. Guijarro C, Egido J. Modulation of the mevalonate pathway: potential mechanisms of vascular protection independent of cholesterol reduction. Cardiovasc Risk Factors. 1997;7:29–33.

15. Gellman J, Ezekowitz MD, Sarembock IJ, Azrin MA, Nochomowitz LE, Lerner E, Haudenschild CC. Effect of lovastatin on intimal hyperplasia after balloon angioplasty: a study in an atherosclerotic hypercholesterolemic rabbit. J Am Coll Cardiol. 1991;17:251–259.[Abstract]

16. Soma MR, Donetti E, Parolini C, Mazzini G, Ferrari C, Fumagalli R, Paoletti R. HMG CoA reductase inhibitors: in vivo effects on carotid intimal thickening in normocholesterolemic rabbits. Arterioscler Thromb.. 1993;13:571–578.[Abstract/Free Full Text]

17. Soma MR, Piliego T, Seregni R, Donetti E, Paoletti R, Fumagalli R. Effect of atorvastatin on intimal carotid thickening induced by perivascular manipulation in normocholesterolemic rabbits. Presented at: 66th Congress of the European Atherosclerosis Society; July, 1996; Firenze, Italy. Abstract.

18. Bocan TM, Mazur MJ, Mueller SB, Brown EQ, Sliskovic DR, O'Brien PM, Creswell MW, Lee H, Uhlendorf PD, Roth BD, Newton RS. Antiatherosclerotic activity of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase in cholesterol-fed rabbits: a biochemical and morphological evaluation. Atherosclerosis. 1994;111:127–142.[Medline] [Order article via Infotrieve]

19. Ross R, Fuster V. The pathogenesis of atherosclerosis. In: Fuster V, Ross R, Topol EJ, eds. Atherosclerosis and Coronary Artery Disease. Philadelphia, Pa: Lippincott-Raven; 1996:441–460.

20. Libby P, Ross R. Cytokines and growth regulatory molecules in atherosclerosis. In: Fuster V, Ross R, Topol EJ, eds. Atherosclerosis and Coronary Artery Disease. Philadelphia, Pa: Lippincott-Raven; 1996:585–594.

21. Hidaka Y, Eda T, Yonemoto M, Kamei T. Inhibition of cultured vascular smooth muscle cell migration by simvastatin (MK-733). Atherosclerosis. 1992;95:87–94.[Medline] [Order article via Infotrieve]

22. Choi KH, Kang SW, Lee SW, Lee HY, Han DS, Kang BS. The effect of lovastatin on proliferation of cultured rat mesangial and aortic smooth muscle cells. Yonsei Med J. 1995;36:251–261.[Medline] [Order article via Infotrieve]

23. Munro E, Patel M, Chan P, Betteridge L, Clunn G, Gallagher K, Hughes A, Schachter M, Wolfe J, Sever P. Inhibition of human vascular smooth muscle cell proliferation by lovastatin: the role of isoprenoid intermediates of cholesterol synthesis. Eur J Clin Invest. 1994;24:766–772.[Medline] [Order article via Infotrieve]

24. Rogler G, Lackner KJ, Schmitz G. Effects of fluvastatin on growth of porcine and human vascular smooth muscle cells in vitro. Am J Cardiol. 1995;76:114A–116A.[Medline] [Order article via Infotrieve]

25. Corsini A, Mazzotti M, Raiteri M, Soma MR, Gabbiani G, Fumagalli R, Paoletti R. Relationship between mevalonate pathway and arterial myocyte proliferation: in vitro studies with inhibitors of HMG-CoA reductase. Atherosclerosis. 1993;101:117–125.[Medline] [Order article via Infotrieve]

26. Pérez Sala D, Collado Escobar D, Mollinedo F. Intracellular alkalinization suppresses lovastatin-induced apoptosis in HL-60 cells through the inactivation of a pH-dependent endonuclease. J Biol Chem. 1995;270:6235–6242.[Abstract/Free Full Text]

27. Borner MM, Myers C, Sartor O, Sei Y, Toko T, Trepel JB, Schneider E. Drug-induced apoptosis is not necessarily dependent on macromolecular synthesis or proliferation in the p53-negative human prostate cancer cell line PC-3. Cancer Res. 1995;55:2122–2128.[Abstract/Free Full Text]

28. Jones KD, Couldwell WT, Hinton DR, Su Y, He S, Anker L, Law RE. Lovastatin induces growth inhibition and apoptosis in human malignant glioma cells. Biochem Biophys Res Commun. 1994;205:1681–1687.[Medline] [Order article via Infotrieve]

29. Patterson SD, Grossman JS, D'Andrea P, Latter GI. Reduced numatrin/B23/nucleophosmin labeling in apoptotic Jurkat T-lymphoblasts. J Biol Chem. 1995;270:9429–9436.[Abstract/Free Full Text]

30. Arends MJ, McGregor AH, Toft NJ, Brown EJ, Wyllie AH. Susceptibility to apoptosis is differentially regulated by c-myc and mutated Ha-ras oncogenes and is associated with endonuclease availability. Br J Cancer. 1993;68:1127–1133.[Medline] [Order article via Infotrieve]

31. Kita T, Brown MS, Goldstein JL. Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in livers of mice treated with mevinolin, a competitive inhibitor of the reductase. J Clin Invest. 1980;66:1094–1100.

32. Owens GK, Loeb A, Gordon D, Thompson MM. Expression of smooth muscle-specific a-isoactin isoform in cultured vascular smooth muscle cells: relationship between growth and cytodifferentiation. J Cell Biol. 1986;102:343–352.[Abstract/Free Full Text]

33. Gottschall AR, Boise LH, Thompson CB, Quintans J. Identification of immunosuppressant-induced apoptosis in a murine B-cell line and its prevention by bcl-x but not by bcl-2. Proc Natl Acad Sci U S A. 1994;91:7350–7354.[Abstract/Free Full Text]

34. González-Cuadrado S, López-Armada MJ, Gómez-Guerrero C, Subirá D, García-Sahuquillo A, Ortiz-González A, Neilson EG, Egido J, Ortiz A. Anti-Fas antibodies induce cytolysis and apoptosis in cultured human mesangial cells. Kidney Int. 1996;49:1064–1070.[Medline] [Order article via Infotrieve]

35. Wu M, Lee H, Bellas RE, Schauer SL, Arsura M, Katz D, FitzGerald MJ, Rothstein TL, Sherr DH, Sonenshein GE. Inhibition of NF-kB/Rel induces apoptosis of murine B cells. EMBO J. 1996;15:4682–4690.[Medline] [Order article via Infotrieve]

36. Vermes I, Haanen C, Steffens Nakken H, Reutelingsperger C. A novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J Immunol Methods. 1995;184:39–51.[Medline] [Order article via Infotrieve]

37. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. J Biochem Physiol. 1959;37:911–917.

38. Parekh AC, Caranto M, Mathur RK, Jung DH. Fractionation of serum cholesterol: a critique. Ann Clin Lab Sci. 1976;6:423–429.[Abstract]

39. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685.[Medline] [Order article via Infotrieve]

40. Majno G, Joris I. Apoptosis, oncosis, and necrosis: an overview of cell death. Am J Pathol. 1996;146:3–15.[Abstract]

41. van Vliet AK, van Thiel GC, Huisman RH, Moshage H, Yap SH, Cohen LH. Different effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors on sterol synthesis in various human cell types. Biochim Biophys Acta. 1995;1254:105–111.[Medline] [Order article via Infotrieve]

42. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature. 1990;343:425–430.[Medline] [Order article via Infotrieve]

43. Grünler J, Ericsson J, Dallner G. Branch-point reactions in the biosynthesis of cholesterol, dolichol, ubiquinone and prenylated proteins. Biochim Biophys Acta. 1994;1212:259–277.[Medline] [Order article via Infotrieve]

44. Quesney-Huneeus V, Wiley MH, Siperstein MD. Essential role for mevalonate synthesis in DNA replication. Proc Natl Acad Sci U S A. 1979;76:5056–5060.[Abstract/Free Full Text]

45. Tsai JC, Jain M, Hsieh CM, Lee WS, Yoshizumi M, Patterson C, Perrella MA, Cooke C, Wang H, Haber E, Schlegel R, Lee ME. Induction of apoptosis by pyrrolidinedithiocarbamate and N-acetylcysteine in vascular smooth muscle cells. J Biol Chem. 1996;271:3667–3670.[Abstract/Free Full Text]

46. Björkerud B, Björkerud S. Contrary effects of lightly and strongly oxidized LDL with potent promotion of growth versus apoptosis on arterial smooth muscle cells, macrophages, and fibroblasts. Arterioscler Thromb Vasc Biol. 1996;16:416–424.[Abstract/Free Full Text]

47. Sinensky M, Lutz R. The prenylation of proteins. Bioessays. 1992;14:25–31.[Medline] [Order article via Infotrieve]

48. Casey PJ, Seabra MC. Protein prenyltransferases. J Biol Chem. 1996;271:5289–5292.[Free Full Text]

49. Denhardt DT. Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signaling. Biochem J. 1996;318:729–747.

50. Gibbs JB, Pompliano DL, Mosser SD, Rands E, Lingham RB, Singh SB, Scolnick EM, Kohl NE, Oliff A. Selective inhibition of farnesyl-protein transferase blocks Ras processing in vivo. J Biol Chem. 1993;268:7617–7620.[Abstract/Free Full Text]

51. Tamanoi F. Inhibitors of Ras farnesyltransferases. Trends Biochem Sci. 1993;18:349–353.[Medline] [Order article via Infotrieve]

52. Kohl NE, Conner MW, Gibbs JB, Graham SL, Hartman GD, Oliff A. Development of inhibitors of protein farnesylation as potential chemotherapeutic agents. J Cell Biochem. 1995;22:145–150.

53. Crowell PL, Chang RR, Ren ZB, Elson CE, Gould MN. Selective inhibition of isoprenylation of 21–26-kDa proteins by the anticarcinogen d-limonene and its metabolites. J Biol Chem. 1991;266:17679–17685.[Abstract/Free Full Text]

54. Hara M, Akasaka K, Akinaga S, Okabe M, Nakano H, Gomez R, Wood D, Uh M, Tamanoi F. Identification of Ras farnesyltransferase inhibitors by microbial screening. Proc Natl Acad Sci U S A. 1993;90:2281–2285.[Abstract/Free Full Text]

55. James GL, Goldstein JL, Brown MS. Polylysine and CVIM sequences of K-RasB dictate specificity of prenylation and confer resistance to benzodiazepine peptidomimetic in vitro. J Biol Chem. 1995;270:6221–6226.[Abstract/Free Full Text]

56. Armstrong SA, Hannah VC, Goldstein JL, Brown MS. CAAX geranylgeranyl transferase transfers farnesyl as efficiently as geranylgeranyl to RhoB. J Biol Chem. 1995;270:7864–7868.[Abstract/Free Full Text]

57. Bobak D, Moorman J, Guanzon A, Gilmer L, Hahn C. Inactivation of the small GTPase Rho disrupts cellular attachment and induces adhesion-dependent and adhesion-independent apoptosis. Oncogene. 1997;15:2179–2189.[Medline] [Order article via Infotrieve]

58. Lebowitz PF, Sakamuro D, Prendergast GC. Farnesyl transferase inhibitors induce apoptosis of Ras-transformed cells denied substratum attachment. Cancer Res. 1997;57:708–713.[Abstract/Free Full Text]

59. Gómez J, Martínez C, Giry M, García A, Rebollo A. Rho prevents apoptosis through Bcl-2 expression: implications for interleukin-2 receptor signal transduction. Eur J Immunol. 1997;27:2793–2799.[Medline] [Order article via Infotrieve]

60. Moorman JP, Bobak DA, Hahn CS. Inactivation of the small GTP binding protein Rho induces multinucleate cell formation and apoptosis in murine T lymphoma EL4. J Immunol. 1996;156:4146–4153.[Abstract]

61. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 1995;270:1326–1331.[Abstract/Free Full Text]

62. Johnson NL, Gardner AM, Diener KM, Langecarter C, Gleavy J, Jarpe MB, Minden A, Karin M, Zon LI, Johnson GL. Signal transduction pathways regulated by mitogen-activated extracellular response kinase kinase kinase induce cell death. J Biol Chem. 1996;271:3229–3237.[Abstract/Free Full Text]

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				U. Schonbeck and P. Libby Inflammation, Immunity, and HMG-CoA Reductase Inhibitors: Statins as Antiinflammatory Agents? Circulation, June 1, 2004; 109(21_suppl_1): II-18 - II-26. [Abstract] [Full Text] [PDF]

				J. Han, X. Zhou, T. Yokoyama, D. P. Hajjar, A. M. Gotto Jr, and A. C. Nicholson Pitavastatin Downregulates Expression of the Macrophage Type B Scavenger Receptor, CD36 Circulation, February 17, 2004; 109(6): 790 - 796. [Abstract] [Full Text] [PDF]

				H. Kawamura, K. Yokote, S. Asaumi, K. Kobayashi, M. Fujimoto, Y. Maezawa, Y. Saito, and S. Mori High Glucose-Induced Upregulation of Osteopontin Is Mediated via Rho/Rho Kinase Pathway in Cultured Rat Aortic Smooth Muscle Cells Arterioscler Thromb Vasc Biol, February 1, 2004; 24(2): 276 - 281. [Abstract] [Full Text]

				C. Li, C. W. Yang, J. H. Park, S. W. Lim, B. K. Sun, J. Y. Jung, S. B. Kim, Y. S. Kim, J. Kim, and B. K. Bang Pravastatin treatment attenuates interstitial inflammation and fibrosis in a rat model of chronic cyclosporine-induced nephropathy Am J Physiol Renal Physiol, January 1, 2004; 286(1): F46 - F57. [Abstract] [Full Text] [PDF]

				V. M. Conraads, J. M. Bosmans, A. J. Schuerwegh, L. S. De Clerck, C. H. Bridts, F. L. Wuyts, W. J. Stevens, and C. J. Vrints Association of lipoproteins with cytokines and cytokine receptors in heart failure patients: Differences between ischaemic versus idiopathic cardiomyopathy Eur. Heart J., December 2, 2003; 24(24): 2221 - 2226. [Abstract] [Full Text] [PDF]

				P. Gargalovic and L. Dory Cellular apoptosis is associated with increased caveolin-1 expression in macrophages J. Lipid Res., September 1, 2003; 44(9): 1622 - 1632. [Abstract] [Full Text] [PDF]

				W.-B. Zhong, C.-Y. Wang, T.-C. Chang, and W.-S. Lee Lovastatin Induces Apoptosis of Anaplastic Thyroid Cancer Cells via Inhibition of Protein Geranylgeranylation and de Novo Protein Synthesis Endocrinology, September 1, 2003; 144(9): 3852 - 3859. [Abstract] [Full Text] [PDF]

				C Abud-Mendoza, H de la Fuente, E Cuevas-Orta, L Baranda, J Cruz-Rizo, and R Gonzalez-Amaro Therapy with statins in patients with refractory rheumatic diseases: a preliminary study Lupus, August 1, 2003; 12(8): 607 - 611. [Abstract] [PDF]

				T. Waehre, J. K. Damas, L. Gullestad, A. M. Holm, T. R. Pedersen, K. E. Arnesen, H. Torsvik, S. S. Froland, A. G. Semb, and P.a. Aukrust Hydroxymethylglutaryl coenzyme a reductase inhibitors down-regulate chemokines and chemokine receptors in patients with coronary artery disease J. Am. Coll. Cardiol., May 7, 2003; 41(9): 1460 - 1467. [Abstract] [Full Text] [PDF]

				J. Genest and T. R. Pedersen Prevention of Cardiovascular Ischemic Events: High-Risk and Secondary Prevention Circulation, April 22, 2003; 107(15): 2059 - 2065. [Full Text] [PDF]

				P. D. Thompson, P. Clarkson, and R. H. Karas Statin-Associated Myopathy JAMA, April 2, 2003; 289(13): 1681 - 1690. [Abstract] [Full Text] [PDF]

				P.O Bonetti, L.O Lerman, C Napoli, and A Lerman Statin effects beyond lipid lowering--are they clinically relevant? Eur. Heart J., February 1, 2003; 24(3): 225 - 248. [Full Text] [PDF]

				T. Maeda, T. Kawane, and N. Horiuchi Statins Augment Vascular Endothelial Growth Factor Expression in Osteoblastic Cells via Inhibition of Protein Prenylation Endocrinology, February 1, 2003; 144(2): 681 - 692. [Abstract] [Full Text] [PDF]

				D. M. Peffley and A. K. Gayen Plant-Derived Monoterpenes Suppress Hamster Kidney Cell 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Synthesis at the Post-Transcriptional Level J. Nutr., January 1, 2003; 133(1): 38 - 44. [Abstract] [Full Text] [PDF]

				W. Dichtl, J. Dulak, M. Frick, H. F. Alber, S. P. Schwarzacher, M. P.S. Ares, J. Nilsson, O. Pachinger, and F. Weidinger HMG-CoA Reductase Inhibitors Regulate Inflammatory Transcription Factors in Human Endothelial and Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, January 1, 2003; 23(1): 58 - 63. [Abstract] [Full Text] [PDF]

				M. R. Mehra, P. A. Uber, K. Vivekananthan, S. Solis, R. L. Scott, M. H. Park, R. V. Milani, and C. J. Lavie Comparative beneficial effects of simvastatin and pravastatin on cardiac allograft rejection and survival J. Am. Coll. Cardiol., November 6, 2002; 40(9): 1609 - 1614. [Abstract] [Full Text] [PDF]

				W. Palinski and C. Napoli Unraveling Pleiotropic Effects of Statins on Plaque Rupture Arterioscler Thromb Vasc Biol, November 1, 2002; 22(11): 1745 - 1750. [Full Text] [PDF]

				C. Kluft, R. Kleemann, and M.P.M. de Maat How best to counteract the enemies? By controlling inflammation in the coronary circulation Eur. Heart J. Suppl., November 1, 2002; 4(suppl_G): G53 - G65. [Abstract] [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]

				F. Gueler, S. Rong, J.-K. Park, A. Fiebeler, J. Menne, M. Elger, D. N. Mueller, F. Hampich, R. Dechend, U. Kunter, et al. Postischemic Acute Renal Failure Is Reduced by Short-Term Statin Treatment in a Rat Model J. Am. Soc. Nephrol., September 1, 2002; 13(9): 2288 - 2298. [Abstract] [Full Text] [PDF]

				W. Palinski and S. Tsimikas Immunomodulatory Effects of Statins: Mechanisms and Potential Impact on Arteriosclerosis J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1673 - 1681. [Abstract] [Full Text] [PDF]

				S. Wassmann, U. Laufs, K. Muller, C. Konkol, K. Ahlbory, A. T. Baumer, W. Linz, M. Bohm, and G. Nickenig Cellular Antioxidant Effects of Atorvastatin In Vitro and In Vivo Arterioscler Thromb Vasc Biol, February 1, 2002; 22(2): 300 - 305. [Abstract] [Full Text] [PDF]

				T. Ichiki, K. Takeda, T. Tokunou, N. Iino, K. Egashira, H. Shimokawa, K. Hirano, H. Kanaide, and A. Takeshita Downregulation of Angiotensin II Type 1 Receptor by Hydrophobic 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors in Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, December 1, 2001; 21(12): 1896 - 1901. [Abstract] [Full Text] [PDF]

				M. Takemoto and J. K. Liao Pleiotropic Effects of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors Arterioscler Thromb Vasc Biol, November 1, 2001; 21(11): 1712 - 1719. [Abstract] [Full Text] [PDF]

				T. Watanabe, M. Yoshizumi, M. Akishita, M. Eto, K. Toba, M. Hashimoto, K. Nagano, Y.-Q. Liang, Y. Ohike, K. Iijima, et al. Induction of Nuclear Orphan Receptor NGFI-B Gene and Apoptosis in Rat Vascular Smooth Muscle Cells Treated With Pyrrolidinedithiocarbamate Arterioscler Thromb Vasc Biol, November 1, 2001; 21(11): 1738 - 1744. [Abstract] [Full Text] [PDF]

				Z. A. Massy and C. Guijarro Statins: effects beyond cholesterol lowering Nephrol. Dial. Transplant., September 1, 2001; 16(9): 1738 - 1741. [Full Text] [PDF]

				B. R. Kwak and F. Mach Statins Inhibit Leukocyte Recruitment: New Evidence for Their Anti-Inflammatory Properties Arterioscler Thromb Vasc Biol, August 1, 2001; 21(8): 1256 - 1258. [Full Text] [PDF]

				R. Patel, S. F. Nagueh, N. Tsybouleva, M. Abdellatif, S. Lutucuta, H. A. Kopelen, M. A. Quinones, W. A. Zoghbi, M. L. Entman, R. Roberts, et al. Simvastatin Induces Regression of Cardiac Hypertrophy and Fibrosis and Improves Cardiac Function in a Transgenic Rabbit Model of Human Hypertrophic Cardiomyopathy Circulation, July 17, 2001; 104(3): 317 - 324. [Abstract] [Full Text] [PDF]

				B. Wong, W. C. Lumma, A. M. Smith, J. T. Sisko, S. D. Wright, and T.-Q. Cai Statins suppress THP-1 cell migration and secretion of matrix metalloproteinase 9 by inhibiting geranylgeranylation J. Leukoc. Biol., June 1, 2001; 69(6): 959 - 962. [Abstract] [Full Text] [PDF]

				K. Yasunari, K. Maeda, M. Minami, and J. Yoshikawa HMG-CoA Reductase Inhibitors Prevent Migration of Human Coronary Smooth Muscle Cells Through Suppression of Increase in Oxidative Stress Arterioscler Thromb Vasc Biol, June 1, 2001; 21(6): 937 - 942. [Abstract] [Full Text] [PDF]

				M. Aikawa and P. Libby Vascular inflammation and activation: new targets for lipid lowering Eur. Heart J. Suppl., May 1, 2001; 3(suppl_B): B3 - B11. [Abstract] [PDF]

				G. P. van Nieuw Amerongen and V. W.M. van Hinsbergh Cytoskeletal Effects of Rho-Like Small Guanine Nucleotide-Binding Proteins in the Vascular System Arterioscler Thromb Vasc Biol, March 1, 2001; 21(3): 300 - 311. [Abstract] [Full Text] [PDF]

				S. Wassmann, U. Laufs, A. T. Bäumer, K. Müller, C. Konkol, H. Sauer, M. Böhm, and G. Nickenig Inhibition of Geranylgeranylation Reduces Angiotensin II-Mediated Free Radical Production in Vascular Smooth Muscle Cells: Involvement of Angiotensin AT1 Receptor Expression and Rac1 GTPase Mol. Pharmacol., March 1, 2001; 59(3): 646 - 654. [Abstract] [Full Text]

				Y. Fukumoto, P. Libby, E. Rabkin, C. C. Hill, M. Enomoto, Y. Hirouchi, M. Shiomi, and M. Aikawa Statins Alter Smooth Muscle Cell Accumulation and Collagen Content in Established Atheroma of Watanabe Heritable Hyperlipidemic Rabbits Circulation, February 20, 2001; 103(7): 993 - 999. [Abstract] [Full Text] [PDF]

				M. Aikawa, E. Rabkin, S. Sugiyama, S. J. Voglic, Y. Fukumoto, Y. Furukawa, M. Shiomi, F. J. Schoen, and P. Libby An HMG-CoA Reductase Inhibitor, Cerivastatin, Suppresses Growth of Macrophages Expressing Matrix Metalloproteinases and Tissue Factor In Vivo and In Vitro Circulation, January 16, 2001; 103(2): 276 - 283. [Abstract] [Full Text] [PDF]

				O. Feron, C. Dessy, J.-P. Desager, and J.-L. Balligand Hydroxy-Methylglutaryl-Coenzyme A Reductase Inhibition Promotes Endothelial Nitric Oxide Synthase Activation Through a Decrease in Caveolin Abundance Circulation, January 2, 2001; 103(1): 113 - 118. [Abstract] [Full Text] [PDF]

				W. Palinsk New Evidence for Beneficial Effects of Statins Unrelated to Lipid Lowering Arterioscler Thromb Vasc Biol, January 1, 2001; 21(1): 3 - 5. [Full Text] [PDF]

				G. P. v. N. Amerongen, M. A. Vermeer, P. Negre-Aminou, J. Lankelma, J. J. Emeis, and V. W. M. van Hinsbergh Simvastatin Improves Disturbed Endothelial Barrier Function Circulation, December 5, 2000; 102(23): 2803 - 2809. [Abstract] [Full Text] [PDF]

				H. Chen, U. Ikeda, M. Shimpo, M. Ikeda, S. Minota, and K. Shimada Fluvastatin Upregulates Inducible Nitric Oxide Synthase Expression in Cytokine-Stimulated Vascular Smooth Muscle Cells Hypertension, December 1, 2000; 36(6): 923 - 928. [Abstract] [Full Text] [PDF]

				Z. Yang, T. Kozai, B. van de Loo, H. Viswambharan, M. Lachat, M. I. Turina, T. Malinski, and T. F. Luscher HMG-CoA reductase inhibition improves endothelial cell function and inhibits smooth muscle cell proliferation in human saphenous veins J. Am. Coll. Cardiol., November 1, 2000; 36(5): 1691 - 1697. [Abstract] [Full Text] [PDF]

				D. E. Marra, T. Simoncini, and J. K. Liao Inhibition of Vascular Smooth Muscle Cell Proliferation by Sodium Salicylate Mediated by Upregulation of p21Waf1 and p27Kip1 Circulation, October 24, 2000; 102(17): 2124 - 2130. [Abstract] [Full Text] [PDF]

				D. C. Hess, A. M. Demchuk, L. M. Brass, and F. M. Yatsu HMG-CoA reductase inhibitors (statins): A promising approach to stroke prevention Neurology, February 22, 2000; 54(4): 790 - 796. [Abstract] [Full Text] [PDF]

				T. Bourcier and P. Libby HMG CoA Reductase Inhibitors Reduce Plasminogen Activator Inhibitor-1 Expression by Human Vascular Smooth Muscle and Endothelial Cells Arterioscler Thromb Vasc Biol, February 1, 2000; 20(2): 556 - 562. [Abstract] [Full Text] [PDF]

				C. Indolfi, A. Cioppa, E. Stabile, E. Di Lorenzo, G. Esposito, A. Pisani, A. Leccia, L. Cavuto, A. M. Stingone, A. Chieffo, et al. Effects of hydroxymethylglutaryl coenzyme A reductase inhibitor simvastatin on smooth muscle cell proliferation in vitro and neointimal formation in vivo after vascular injury J. Am. Coll. Cardiol., January 1, 2000; 35(1): 214 - 221. [Abstract] [Full Text] [PDF]

				A. H. Wagner, T. Kohler, U. Ruckschloss, I. Just, and M. Hecker Improvement of Nitric Oxide-Dependent Vasodilatation by HMG-CoA Reductase Inhibitors Through Attenuation of Endothelial Superoxide Anion Formation Arterioscler Thromb Vasc Biol, January 1, 2000; 20(1): 61 - 69. [Abstract] [Full Text] [PDF]

				A. Faggiotto and R. Paoletti Statins and Blockers of the Renin-Angiotensin System : Vascular Protection Beyond Their Primary Mode of Action Hypertension, October 1, 1999; 34(4): 987 - 996. [Abstract] [Full Text] [PDF]

				R. H. WEISS, A. RAMIREZ, and A. JOO Short-Term Pravastatin Mediates Growth Inhibition and Apoptosis, Independently of Ras, via the Signaling Proteins p27Kip1 and PI3 Kinase J. Am. Soc. Nephrol., September 1, 1999; 10(9): 1880 - 1890. [Abstract] [Full Text]

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