138(2), 446–456 2014 doi: 10.1093/toxsci/kfu011 Advance Access publication January 21, 2014

TOXICOLOGICAL SCIENCES

Dysfunction of Vascular Smooth Muscle and Vascular Remodeling by Simvastatin Seojin Kang,* Hyang-Hwa Woo,* Keunyoung Kim,* Kyung-Min Lim,† Ji-Yoon Noh,* Moo-Yeol Lee,‡ Young Min Bae,§ Ok-Nam Bae,¶ and Jin-Ho Chung*,1 *College of Pharmacy, Seoul National University, Seoul 151-742, Korea; †College of Pharmacy, Ewha Womans Universtiy, Seoul 120-750, Korea; ‡College of Pharmacy, Dongguk University, Goyang 410-820, Korea; §School of Medicine, Konkuk University, Choongju 380-701, Korea; and ¶College of Pharmacy, Hanyang University, Ansan 426-791, Korea whom correspondence should be addressed at College of Pharmacy, Seoul National University, Shinrim-dong San 56-1, Seoul 151-742, Korea. Fax: +82-2-885-4157. E-mail: [email protected]. Received October 28, 2013; accepted January 4, 2014

Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, are widely prescribed for hypercholesterolemia. With the increasing use of statins, numerous reports demonstrated that statins can cause damage to skeletal muscles. However, the toxicities of statins on vascular smooth muscle, which are essential to cardiovascular homeostasis, have not been previously described. Here, we examined the effects of simvastatin on the contractile function and the integrity of vascular smooth muscle in isolated rat thoracic aortic rings, primary cultured vascular smooth muscle cells (VSMCs) in vitro and rats in vivo. In aortic rings, simvastatin suppressed the normal agonist-induced contractile responses in timeand concentration-dependent manners (0.86 g ± 0.11 at 10␮M simvastatin for 24 h compared with 1.89 g ± 0.11 at control). The suppression persisted in the endothelium-denuded aortic rings and was irreversible even after wash-out of simvastatin. Simvastatin suppressed the contraction induced by Bay K8644, an activator of voltage-operated Ca2+ channel (VOCC) in rat aortic rings and abolished agonist-induced intracellular Ca2+ increase in VSMCs. The simvastatin-induced contractile dysfunction was reversed by the supplementation of mevalonate and geranylgeranylpyrophosphate, precursors for protein isoprenylation. Consistently, activation of RhoA, a representative isoprenylated protein, was disrupted by simvastatin in VSMCs and RhoA-mediated phosphorylation of MYPT1 and CPI-17, and tonic tension were also suppressed. Notably, prolonged treatment of simvastatin up to 48 h induced apoptosis of vascular smooth muscle in aortic rings. Most importantly, simvastatin treatment in vivo significantly attenuated the agonistinduced vasoconstriction in rats ex vivo and induced a decrease in luminal area of the vascular wall. Collectively, these results demonstrate that simvastatin can impair the normal vascular contractility by disturbing Ca2+ influx and RhoA activity, ultimately leading to apoptosis and structural remodeling. Key words: smooth muscle dysfunction; simvastatin; Ca2+ influx; RhoA; apoptosis; vascular remodeling.

Statins represent the most widely used lipid-lowering agents in the world (Taylor et al., 2011). Statins block 3-hydroxy-3-

methylglutaryl-coenzyme A (HMG-CoA) reductase which catalyzes the conversion of HMG-CoA to mevalonate, a common precursor for the syntheses of cholesterol and ubiquinone. It is an early and rate-limiting step in the biosynthesis of cholesterol and through its blockade statins can effectively lower total and low-density lipoprotein (LDL) cholesterol in blood, ultimately alleviating hypercholesterolemia, which is critical to the primary and secondary prevention of life-threatening atherosclerosis and coronary heart diseases (Stancu and Sima, 2001). In addition to cholesterol-lowering effects, statins have many pleiotropic effects on cardiovascular physiology that include the modulation of vascular redox signaling and nitric oxide bioavailability, stabilization of atherosclerotic plaques, attenuation of inflammation and oxidative stress, and the inhibition of thrombogenic responses in the vascular wall (Antonopoulos et al., 2012; Bellosta et al., 1998; Liao and Laufs, 2005). Indeed, statins modulate the production of many endogenous products other than cholesterol, such as coenzyme Q10, heme-A, and isoprenylated proteins that play central roles in cell biology and human physiology. Accordingly, alteration of these metabolites may produce benefits as well as toxicities. Especially, with the increasing use of statins, reports of adverse effects are growing such as hepatotoxicity, neuropathy, gastrointestinal disturbances, and myotoxicity (Golomb and Evans, 2008). Of these, myotoxicity is the most common and severe. The incidence rate of myotoxicity reaches 1–7% of statin-treated patients (Thompson et al., 2003). Myotoxicity of statins is manifested in diverse forms and to a varying degree of severities from mild myalgia or muscle weakness to lifethreatening rhabdomyolysis (Joy and Hegele, 2009). Although rare in occurrence, rhabdomyolysis, a massive and precipitous destruction of muscle fibers, results in the release of myoglobin into blood stream and subsequently leads to kidney failure and deaths. Pathophysiological mechanism underlying the statininduced myotoxicity has not been fully clarified (ChristopherStine, 2006) but the involvement of cholesterol-lowering activ-

 C The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected]

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tion of mevalonate or geranylgeranylpyrophosphate, precursors of protein prenylation, suggesting that insufficient prenylation may have caused these events. We believe that this study may enlighten the potential vascular toxicity of statins, bringing to attention the risk of inadvertent use of statins.

MATERIALS AND METHODS

Reagents. The following chemicals were purchased from Sigma Chemical Co. (St Louis, MO): Simvastatin, phenylephrine (PE), endothelin-1, lysophosphatidylcholine (LPC), serotonin creatinine sulfate, acetylcholine, geranylgeraniol (GGOH), mevalonate, squalene, nifedipine, bromoenol lactone, thapsigargin, (-)-(S)-Bay K8644, and dimethyl sulfoxide (DMSO). Fura-2/AM was obtained from Molecular Probes (Eugene, OR) and Dulbecco’s modified Eagle medium (DMEM), minimum essential media (MEM), penicillin/streptomycin and fetal bovine serum used in cell culture were purchased from Gibco Co. (Carlsbad, CA). All other reagents used were of the highest purity available. Animals. Eight-week-old male Sprague Dawley rats (SamTako, Korea) weighing 250–350 g were used in experiments except for the primary smooth muscle cell culture using 5-weekold animals weighing 150–170 g. Before the experiments, the animals were acclimated for 1 week under controlled laboratory conditions of temperature at 22◦ C ± 2◦ C and humidity at 55 ± 5% with 10 h light/14 h dark cycle. Normal diet (Cargill Agri Purina, Inc., Korea) and water were provided ad libitum. All protocols were approved by the Ethics Committee of the Animal Service Center at Seoul National University. More than four animals were used in each in vitro experiments. For the measurement of ex vivo vascular contraction four animals per each group were used and eight animals per each group were used for in vivo vascular remodeling. Measurement of vasoconstriction in isolated aortic rings. Rats were sacrificed by decapitation and then exsanguinated. The thoracic aorta was carefully isolated and cut into ring segments and the aortic rings were placed in MEM immediately. Aortic rings without endothelium were prepared by gently rubbing off the intimal surface of the aortic rings with a wooden stick. To investigate the effect of simvastatin on vasoconstriction, we incubated the aortic rings with simvastatin (0.1, 1, or 10␮M) or vehicle (DMSO) in MEM supplemented with 100 U/ml penicillin and 100 ␮g/ml streptomycin in a 95% O2 /5% CO2 incubator for 24 h at 37◦ C. Measurement of vasoconstriction of aortic rings was performed as reported previously (Lee et al., 2010). The aortic rings were mounted on organ baths filled with Krebs-Ringer solution (KR solution; 115.5mM NaCl, 4.6mM KCl, 1.2mM KH2 PO4 , 1.2mM MgSO4 , 2.5mM CaCl2 , 25.0mM NaHCO3 , and 11.1mM glucose, pH 7.4). KR solution was continuously gassed with 95% O2 /5% CO2 and

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ity and resultant alteration of the cellular membrane fluidity has been suggested to play a role (Hodel, 2002; Sirvent et al., 2008). Perturbation of the membrane fluidity may affect the activities of ion channels such as sodium, potassium, and chloride channels that are critical in the maintenance of membrane excitability of muscle (Bastiaanse et al., 1997). Calcium channels that play an essential role in contraction and other fundamental functions of muscle are also affected by statins (Alvarez de Sotomayor et al., 2001; Seto et al., 2007). The disruption of calcium homeostasis by statins is suggested to be from the impairment of mitochondrial respiration or the disturbance of intracellular calcium signaling (Mohaupt et al., 2009). Insufficient protein prenylation from the blockade of mevalonate/farnesylpyrophosphate/ geranylgeranylpyrophosphate axis is also believed to be an important contributor to the statin-induced myotoxicity (Dirks and Jones, 2006). Prenylation is a key post-translational modification step for the functional maturation of vital proteins in the muscle such as Rho, Ras, Rac, and Rab that regulate ion homeostasis, muscle contraction, cellular signaling, differentiation, proliferation, and apoptosis (Berzat et al., 2005; Zhang and Casey, 1996). Insufficient protein prenylation results in the inactivation of these proteins and may cause a variety of adverse effects of biological significance such as insufficient phosphorylation of myosin light chain (MLC), malfunction of membrane excitability (Bayguinov et al., 2011), and premature cell deaths. The most frequent target of statin-induced myotoxicity is skeletal muscle but toxicity to cardiac muscle tissue has also been reported (Rabkin and Kong, 2003) suggesting that muscle tissues and molecular targets concerning the muscular functions may be broadly affected by statins. Vascular smooth muscle, a major category of muscles, is essential to the distribution of blood to tissues and cells through maintaining adequate blood pressure (Fisher, 2010). Vascular smooth muscle contains sodium, chloride, potassium, and calcium channels that are essential in controlling muscle contraction. Importantly, blood vessels express high level of HMG-CoA reductase (Seto et al., 2007) and small GTPase RhoA, one of the most well-known target proteins for prenylation, plays critical roles in the modulation of ion homeostasis, calcium sensitization, and tonic tension. In this regard, it is highly probable that statins might affect normal function or structural integrity of vascular smooth muscle through impairment of RhoA activation and vascular smooth muscle also could be a target of myotoxicity by statins as well as skeletal muscle. In this study, we found that simvastatin, one of the most widely used statins, could irreversibly impair the vasoconstrictory responses to physiological agonists in isolated rat aortic rings. Tonic tension, maintenance of generated tension, were also impaired by simvastatin and agonist-induced calcium influx was suppressed in vascular smooth muscle cells (VSMCs). Most notably, we demonstrated that after repeated oral exposure to simvastatin, the structure of blood vessel was altered in rats in vivo. These effects could be rescued by the supplementa-

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Measurement of lactate dehydrogenase leakage. To examine the nonspecific cytotoxicity of simvastatin, the extent of lactate dehydrogenase (LDH) leakage was measured using a previously described method (Suenaga and Kamata, 1999). Aortic rings without endothelium were incubated with simvastatin (0.1, 1 or 10␮M) or vehicle for 24 h. After the incubation period, the incubation solution was removed and put into Tris-EDTA buffer (56mM Tris, 5.6mM EDTA, pH 7.4) containing 0.17mM NADH and then incubated for further 10 min at 37◦ C. The incubation solution was supplemented with prewarmed pyruvate (14mM). LDH in the solution was determined by measuring absorbance at 339 nm using UV spectrophotometer (Shimadzu, Japan). Four independent experiments were performed. Measurement of cytosolic Ca2+ changes in primary rat smooth muscle cells. The level of cytosolic calcium was measured with fluorometric method employing fura-2 and digital imaging as described previously (Lee et al., 2006). After the endothelium and adventitia were removed, aortic rings were chopped finely and smooth muscle cells were liberated from the tissue in DMEM containing 300 units of collagenase type 2 and 3 units of elastase (Worthington Biochemical Corp., Lakewood, NJ) at 37◦ C for 1 h. After seeding on coverslips in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 ␮g/ml streptomycin, smooth muscle cells were grown in at 37◦ C in a 95% O2 /5% CO2 incubator and then treated with simvastatin for 24 h. To load fura-2, cells were incubated in phosphate salt solution (PSS; 140mM NaCl, 5.0mM KCl, 1.4mM MgCl2 , 1.2mM NaH2 PO4 , 10mM

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 5mM NaHCO3 , 1.8mM CaCl2 , and 11.5mM glucose, pH 7.4) containing 1␮M fura-2/AM and 1% bovine serum albumin for 60 min. Coverslips were mounted in a superfusion chamber on the microscope stage and were superfused with PSS (2 ml/min). All experiments were performed at 33◦ C. Cells were imaged with a Nikon Eclipse Ti-U inverted microscope equipped with an S Fluor 40X (N.A. 1.30, oil) objective lens (Nikon, Melville, NY) and an Evolve EMCCD Camera (Photometrics, Tucson, AZ). Illumination was provided by a Sutter DG-4 filter changer (Sutter Instruments, Novato, CA). Excitation and emission wavelengths used for fura-2 were 340/380 and 535 nm, respectively. Images were acquired and analyzed with a Meta Imaging System (Molecular Devices, West Chester, PA). Cytosolic Ca2+ changes were measured from 40–50 cells obtained in three coverslips. Determination of simvastatin effects on intracellular and extracellular Ca2+ pathways. To investigate the effect of simvastatin on the contraction induced by the influx of extracellular Ca2+ , Bay K8644, an L-type Ca2+ channel opener, was used. Aortic rings treated with simvastatin (0.1, 1, or 10␮M) for 24 h were mounted on an organ bath. Bay K8644 was added cumulatively in 15mM K+ buffer solution (105mM NaCl, 15.0mM KCl, 1.2mM KH2 PO4, 1.2mM MgSO4 , 2.5mM CaCl2 , 25.0mM NaHCO3 , 0.026mM EDTA, and 11.1mM glucose, pH 7.4) to initiate vasoconstriction. To examine the effect of simvastatin on the contraction mediated by the release of the intracellular Ca2+ store, vasoconstriction was induced in Ca2+ -free condition. After treated with simvastatin for 24 h, aortic rings were mounted on an organ bath filled with Ca2+ -free KR solution (120mM NaCl, 5.9mM KCl, 1.2mM NaH2 PO4 , 1.2mM MgCl2 , 25mM NaHCO3 , 2mM EGTA, and 11.1mM glucose, pH 7.4). For selective activation of store-operated Ca2+ channel (SOCC), the aortic rings without an endothelium were pre-treated with 1␮M thapsigargin (an inhibitor of sarcoplasmic reticulum Ca2+ ATPase) in Ca2+ -free KR solution to deplete the intracellular Ca2+ store. Aortic rings were then treated with simvastatin or vehicle for 4 h, and the bath solution was exchanged into KR solution containing 2.5mM Ca2+ to induce SOCC-mediated contraction. Transient receptor potential cation channel inhibitor, bromoenol lactone, was used as positive control. For each experiment 4–7 independent replicates were performed. Determination of RhoA activation. RhoA activity was measured using a commercially available Rho activation assay kit (Cytoskeleton, Inc., Denver, CO) as instructed by the manufacturer. Primary cultured rat VSMCs (80–90% confluent) grown in T-25 flasks were incubated with simvastatin 10␮M and additional GGOH for 1 h and then treated with 10−5 M PE for 10 min. The cells were harvested on ice in lysis buffer containing protease inhibitor cocktail (Thermo Scientific, Rockford, IL). Cell debris was removed by a 15-min centrifugation at 9800 × g, and the supernatant was incubated with rhotekin-RBD beads

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maintained at 37◦ C. The rings were stretched gradually to an optimal resting tension at 2 g and equilibrated for 30 min. The change in tension was measured isometrically with Grass FT03 force transducers (Grass Instrument Co., Quincy, MA) and recorded using the AcqKnowledge III computer program (BIOPAC Systems, Inc., Goleta, CA). The vasoconstriction was induced by cumulative addition of PE (up to 10−5 M), serotonin (up to 10−4 M), or endothelin-1 (up to 10−6 M) within 30 min to obtain concentration-contraction curves. In order to examine whether simvastatin affects the stage of tension maintenance, that is, the tonic tension, simvastatin was treated after the induction of vasoconstriction by PE and continuously incubated for 2 h. To examine the irreversibility of simvastatin effects on vasoconstriction, PE-induced contraction was measured after treatment with simvastatin for 24 h in aortic rings without endothelium. The irreversibility of simvastatin effects on PE-induced contraction was evaluated 1 h after washing out of simvastatin-containing KR buffer in an organ bath. The effect of nifedipine (1␮M, 24 hr) was compared with the effect of simvastatin. Effect of additional supplement of HMG-CoA reductase intermediates was measured after GGOH (30␮M), mevalonate (100␮M), or squalene (100mM) was treated with simvastatin (10␮M) to aortic rings for 24 h. For each experiment 4–7 independent replicates were performed.

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specifically recognizing active RhoA for 1 h at 4◦ C. Beads were washed, and the precipitated RhoA was detected by Western blotting using specific monoclonal antibody against RhoA. Four independent experiments were performed.

Terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick-end-labeling staining of aortic rings and histological assessment. After aortic rings were placed in MEM containing 100 U/ml penicillin and 100 ␮g/ml streptomycin, simvastatin, GGOH, or vehicle was treated. They were then incubated in a 95% O2 /5% CO2 incubator for 24 h at 37◦ C. After incubation, the aortic rings were fixed in a buffered formalin solution (10%) and embedded in paraffin. Terminal deoxynucleotidyl transferase (TdT) mediated deoxyuridine triphosphate (dUTP) nick-end-labeling (TUNEL) assays were done using a commercial kit according to the manufacturer’s instructions (Chemicon International, Temecula, CA). The embedded tissue was sectioned at a thickness of 4 ␮m and was placed on an adhesive slide. The section was deparaffinized by washing with xylene following serial dehydration with ethanol (100, 95, 80, and 70%). Dehydrated sections were treated with 0.3% H2 O2 to quench endogenous peroxidase activity followed by 20 ␮g/ml DNase-free Proteinase K to retrieve antigenic epitopes. Subsequently, the sections were treated with TdT enzyme reagent for 1 h at 37◦ C to label-free 3 -OH termini with digoxigenin-dUTP. To detect incorporated digoxigenin-conjugated nucleotides, horseradish peroxidase–conjugated anti-digoxigenin antibody and 3,3 -diaminobenzidine (DAB) were used. Sections were treated with anti-digoxigenin-peroxidase for 30 min at room temperature, and this was followed by DAB development. The sections were counterstained with Mayer’s hematoxylin. Dehydrated sections were then cleaned in xylene and mounted. For assessment of TUNEL-positive cells, the numbers of total cell nuclei and positive cell nuclei were counted in four fields

for each specimen and the percentages of positive cell nuclei were calculated. Determination of caspase-3 activity. Primary cultured rat VSMCs (80–90% confluent) grown in T-25 flasks were incubated with simvastatin and additional GGOH for 48 h and then harvested on ice in lysis buffer containing protease/phosphatase inhibitor cocktail (Thermo Scientific). Cell debris was removed by a 15-min centrifugation at 9800 × g. The supernatant was suspended in Laemmli sample buffer (Bio-Rad) and boiled for 5 min at 99◦ C. Active caspase-3 and procaspase-3 were detected by Western blot analysis using anti-caspase-3 (Cell Signaling Technology, Inc.) antibody. In vivo evaluation of vascular remodeling by repeated treatment of simvastatin. Simvastatin (80 mg/kg) or vehicle (10% ethanol in saline) was administered orally to eight male SD rats per each treatment group once daily for 7 days. The animals were euthanized by ethylether to obtain the thoracic aorta through formalin perfusion. The entire thoracic aorta were obtained and fixed in a buffered formalin solution (10%) and embedded in paraffin. Horizontal ring sections were carefully obtained without folds at identical points in the aorta, and hematoxylin and eosin staining was used for microscopic observation. The area of the lumen and the medial layer of the blood vessel were measured and analyzed using Image J program. For the estimation of the luminal area, the circumference of the lumen was obtained and used to calculate the area assuming a circular structure, according to the method described previously (Harmon et al., 2000). The medial area was calculated the area defined by the luminal surface and the outer boundary of the aorta. Statistical analysis. All the data are represented by mean ± SEM, and the data were subjected to one-way analysis of variance followed by Duncan’s multiple ranged tests to determine which means were significantly different from the control. Statistical analysis was performed using SPSS software (Chicago, IL). In all cases, p value of < 0.05 was used to determine significance. RESULTS

We examined if simvastatin can affect the agonist-induced vasoconstriction, a major function of vascular smooth muscle. After simvastatin was pre-treated to isolated rat aortic rings, PE, adrenergic ␣-agonist, was cumulatively added to induce vasoconstriction. Although simvastatin had no effect on basal tone (data not shown), it significantly inhibited PE-induced vasoconstriction of rat aortic rings in concentration- and time-dependent manners (Fig. 1A and B). Similar patterns of inhibition could be observed in the contraction induced by other contractile agonists that include serotonin and endothelin-1 (Fig. 1C), suggesting that simvastatin affects common contractile pathways

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Measurement of MYPT1 and CPI-17 phosphorylation and Western blot analysis. After treated with simvastatin and additional GGOH for 24 h, aortic rings without endothelium were immediately frozen by liquid nitrogen. Frozen aortic rings were then homogenized in RIPA buffer containing protease/phosphatase inhibitor cocktail and were centrifuged at 9800 × g for 15 min at 4◦ C. The supernatant was suspended in Laemmli sample buffer (Bio-Rad, Hercules, CA) and boiled for 5 min at 99◦ C. Protein extracts were electrophoresed in a polyacrylamide mini-slab gel and then transferred onto PVDF membranes in Tris/glycine buffer (25mM Tris, 192mM glycine) containing 20% methanol. The extent of phosphorylation was measured with immunoblotting using anti-phospho-MYPT1, anti-MYPT1 (Cell Signaling Technology, Inc., Danvers, MA), anti-phospho-CPI-17, and anti-CPI-17 (Abcam, UK) antibodies respectively. Immunoreactive bands were visualized by horseradish-peroxidase-conjugated secondary antibody and a Pico chemiluminescence kit (Thermo Scientific).

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shared by diverse agonists rather than specific receptor activation. Attenuation of agonist-induced vasoconstriction can be caused either from vasorelaxation mediated by endothelial cells or inhibition of vascular smooth muscle contraction. To identify the target for the anti-contractile effects of simvastatin, endothelium-denuded aortic ring system was employed. As seen in Figure 1D, the suppression of PE-induced vasoconstriction was retained in endothelium-denuded aortic rings, suggesting that contractile machinery of vascular smooth muscle is affected directly by simvastatin. Vasoconstriction of vascular smooth muscle develops in two distinct stages, i.e., the initial generation of tension (phasic tension) and the maintenance of tension (tonic tension). To examine the effects of simvastatin on tonic tension, simvastatin was post-treated after tension was generated with PE. As shown in Figure 1E, simvastatin significantly disrupted tonic tension, reflecting that both stages of vasoconstriction were affected. Nonspecific tissue cytotoxicity was not involved, as determined by the absence of LDH leakage up to 10␮M simvastatin (Fig. 1F).

Of particular note, the suppression of vasoconstriction by simvastatin was not recovered after simvastatin was washed off (Fig. 2A). Even after longer recovery times, contractile responses were not regained (Fig. 2B) which was in a good contrast to the reversible effects of nifedipine, an antihypertensive drug (Fig. 2C), suggesting that irreversible alteration of contractile machinery and permanent dysfunction of smooth muscle has been caused by simvastatin. We tried to elucidate the mechanism underlying simvastatininduced smooth muscle dysfunction. Contraction is initiated by the agonist-induced intracellular calcium increase. Simvastatin significantly blocked agonist-induced intracellular calcium increase (Fig. 3A). Agonist-induced calcium signal is provisioned by extracellular calcium influx through voltageoperated calcium channel (VOCC), store-operated calcium channel (SOCC), or release from intracellular calcium store (ICS). To narrow down the mechanism of calcium blockade, effects of simvastatin on the vasoconstriction primed by respective activation of VOCC, SOCC, or ICS was examined.

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FIG. 1. Effect of simvastatin on agonist-induced vasoconstriction in rat aortic rings. After simvastatin (SIM) was treated to aortic rings with an intact endothelium for 24 h, (A) concentration- and (B) time-dependent effects (at 10␮M) on PE-induced vasoconstriction were obtained. (C) Serotonin or endothelin-1 was used to induce vasoconstriction after treatment with simvastatin for 24 h. (D) Effect of simvastatin in aortic rings without endothelium was also observed. After simvastatin was treated to endothelium-denuded aortic rings for 24 h, concentration-dependent effect on PE-induced vasoconstriction was obtained. (E) Simvastatin (10␮M) was post-treated after the initiation of the contraction by PE (10−5 M) and effect of post-treatment of simvastatin on contraction was observed. (F) Nonspecific cytotoxicity was determined by LDH leakage. LPC (10−4 M) was used as a positive control. Values are mean ± SEM of more than four independent experiments. * represents significant differences from the controls (p < 0.05).

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Although little effect could be found for intracellular-calciummediated (Fig. 3C) or SOCC-mediated vasoconstriction (Fig. 3D), significant inhibition was observed for VOCC-mediated vasoconstriction (Fig. 3B), suggesting that simvastatin blocks VOCC-mediated calcium increase. Further confirming this, intracellular calcium increases induced by Bay K8644 and high potassium-induced membrane depolarization were significantly attenuated by simvastatin pre-treatment (Fig. 3E). In addition, inhibition of Bay K8644 induced contraction persisted even after the wash-out of simvastatin (Fig. 3F). Simvastatin inhibits HMG-CoA reductase which produces mevalonate, a common precursor for cholesterol (via squalene) and geranylgeranylpyrophosphate. Especially, geranylgeranylpyrophosphate is essential in the functional maturation of isoprenylated proteins like RhoA (Fig. 4A). To investigate the mechanism underlying the contractile dysfunction by simvastatin, we examined the effects of cotreatment with mevalonate, geranylgeraniol (GGOH), and squalene. As shown in Figures 4B and C, suppression of PE- and Bay K8644-induced vasoconstriction by simvastatin was fully reversed by mevalonate and GGOH whereas squalene had no effect, reflecting that insufficient protein prenylation is central to the smooth muscle dysfunction by simvastatin. Indeed, PE-induced acti-

vation of RhoA, a representative isoprenylated small GTPase protein, was significantly suppressed by simvastatin (Fig. 4D). RhoA is also critical for tonic tension by sustaining MLC phosphorylation through MYPT1- and CPI-17-mediated MLC phosphatase inhibition (Fig. 4A). Consistently, simvastatin decreased MYPT1 and CPI-17 phosphorylation (Figs. 4E and F), which was reversed by the supplementation of GGOH. Dysfunction may eventually affect the structural integrity of normal tissue. As expected, prolonged treatment of simvastatin induced apoptosis of smooth muscle as can be determined by TUNEL staining of blood vessel in vitro (Fig. 5A) and activation of caspase-3 from procaspase-3 in primary smooth muscle cells (Fig. 5B), strongly supporting that the anti-contractile effects of simvastatin must not be regarded as therapeutic effects. To investigate the in vivo consequence of the simvastatin-induced vascular smooth muscle dysfunction, rats were orally administered with simvastatin for 7 days and contractile responses of aortic rings to PE were evaluated ex vivo. As shown in Figure 6A, repeated treatment of simvastatin attenuated the contractile responses to PE whereas supplementation of GGOH lessened it substantially. More importantly, the vascular structure was altered by simvastatin treatment as observed by reduced area of lumen and media (Fig. 6B). This vascular remodeling was

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FIG. 2. Irreversibility of simvastatin effects on PE-induced vasoconstriction. (A) After treatment with simvastatin for 24 h in aortic rings without endothelium, PE-induced contraction was measured, I. The irreversibility of simvastatin effects on PE-induced contraction was evaluated 1 h after washing out of simvastatincontaining KR buffer in an organ bath, II. The effect of simvastatin (10␮M, 24 h) was measured 1 or 4 h after washing out of simvastatin-containing KR buffer (B) and the effect of nifedipine, an antihypertensive drug (1␮M, 24 h), was compared (C). Values are mean ± SEM of more than four independent experiments. * represents significant differences from the controls (p < 0.05).

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markedly attenuated by the supplementation of GGOH confirming that impairment of mevalonate/GGOH production and isoprenylation by simvastatin may have induced vascular smooth muscle dysfunction and structural alteration of blood vessels.

DISCUSSION

Here we demonstrated that the myotoxicity of simvastatin is not limited to skeletal or cardiac muscles but also affects vascular smooth muscle. Simvastatin-induced vascular smooth muscle dysfunction through the suppression of VOCC-mediated intracellular calcium increase and the disruption of RhoA/RhoA kinase (ROCK) regulated tonic tension that eventually led to functional abnormality, apoptosis, and a structural remodeling of blood vessels after prolonged exposure in vivo. Vascular smooth muscle dysfunction and vascular remodeling may parallel the myotoxicity of statins observed in skeletal muscle which is manifested as muscle weakness and rhabdomyolysis. Simvastatin-induced vascular smooth muscle dysfunction may have profound consequences via altering hemodynamic homeostasis that, we believe, warrants further study in human.

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FIG. 3. Effect of simvastatin on Ca2+ mobilization and vasoconstriction. (A) Inhibition of serotonin (10−7 M) induced calcium increase by simvastatin (10␮M, 24 h) was observed in primary cultured rat VSMCs using fura-2/AM. (B) After simvastatin was treated to aortic rings without endothelium, vasoconstriction was initiated with an addition of Bay K8644 (10−7 M), an L-type Ca2+ channel opener. Nifedipine (NIF; 1␮M), a voltage-sensitive L-type Ca2+ channel blocker, was used as a positive control. (C) Effects of simvastatin on PE (10−5 M) induced vasoconstriction in Ca2+ -free buffer were determined. (D) Simvastatin was treated to aortic rings without endothelium to measure SOCCmediated contraction, as described in Materials and Methods. Bromoenol lactone (BEL; 25␮M) was used as a positive control. (E) Inhibitions of Bay K8644 and 75mM K+ -induced calcium increase by simvastatin in primary VSMCs were also observed using fura-2/AM. (F) The irreversible effect of simvastatin on Bay K8644 (10−7 M) induced contraction was also investigated. After treatment with simvastatin for 24 h in aortic rings without endothelium, Bay K8644induced contraction was measured at “I.” The irreversible effect was measured 1 h after washing out of simvastatin at “II.” Values are mean ± SEM of more than four independent experiments. * represents significant differences from the controls (p < 0.05).

Due to pleiotropic effects of statins on cardiovascular tissues, statins are widely prescribed to patients with diverse cardiovascular diseases. With the expiry of patents of major statins including simvastatin and atorvastatin, prices of statins have declined drastically. Big pharmaceutical companies like Merck or Pfizer are endeavoring to switch statins to over-the-counter (OTC) drugs to compensate the financial loss. Indeed, in United Kingdom, 10 mg dose of simvastatin has been approved for OTC sales in 2004 (Gotto, 2006). These trends may substantially increase the chance of exceedingly high and long-term use of statins and consequently elevate the risk of statin-associated adverse effects that include the vascular toxicity. We also could observe that atorvastatin, another blockbuster statin, suppresses vascular constriction suggesting that smooth muscle dysfunction may be a class effect of statins. Although it is important to lower the pathologically high level of lipid and serum cholesterol for the prevention of atherogenesis and cardiac complications, the potentially deleterious effects of statin abuse on the normal vascular structure and hemodynamic homeostasis must not be ignored. Here we showed that simvastatin caused the malfunction of VOCC and the perturbation of tonic tension. The alteration of intracellular calcium homeostasis in VSMCs and inhibition of vasoconstriction by lipophilic statins like atorvastatin and simvastatin was already discovered by several groups (Alvarez de Sotomayor et al., 2001; Seto et al., 2007; Tesfamariam et al., 1999). However, these effects were considered beneficial because they may lower the blood pressure in the hypertensive patients. In this context, these studies employed or assumed pathological settings like atherosclerosis or hypertension and interpreted the effects of statin as beneficial, paying little attention to the potential risk of collateral damages to the normal vasculature. In the present study, we could observe that the suppression of vasoconstriction by simvastatin was irreversible, which was in a clear contrast to the reversible effects of nifedipine. More importantly, prolonged incubation of simvastatin induced apoptosis in smooth muscle and repeated oral administration of simvastatin altered the vascular structure in rats in vivo, demonstrating that it may cause functional and structural alteration of normal vasculature. This finding may be in line with the previous reports that raise questions on the benefit of statins on the prevention of cardiovascular morbidity and mortality especially when LDL-cholesterol level is not high (Byington et al., 1995; Sacks et al., 1996), suggesting that the pleiotropic effects of simvastatin on vascular tissues must be fully substantiated through the careful examination of the effects of statin on normal tissues. PE- and serotonin-induced calcium entries were attenuated by simvastatin. Moreover, contraction induced by a VOCC activator, Bay K8644, and high potassium was inhibited by simvastatin, suggesting that intracellular calcium increases following agonist binding or membrane depolarization are affected by simvastatin. Lipophilic simvastatin can nonspecifically intercalate into the membrane lipid bilayer, perturbing membrane integrity and ion channel activities (Yada et al.,

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1999). However, we could observe that inhibition of vasoconstriction by simvastatin was attenuated by the addition of mevalonate/geranylgeraniol (GGOH), indicating that insufficient protein prenylation and inactivation of prenylated proteins like RhoA may be more important. Involvement of RhoA in simvastatin-inhibited calcium increases has been suggested previously (Bergdahl et al., 2003; Ng et al., 1994; Tesfamariam et al., 1999). RhoA/ROCK pathway is involved in the regulation of Ca2+ and other cation ion channels (Pochynyuk et al., 2006; Villalba et al., 2008). Recently, Bayguinov et al. reported that RhoA inhibitors attenuate contraction of murine colonic smooth muscle through impairing nonspecific cation channel, which is critical to membrane excitation and voltage-induced calcium entry (Bayguinov et al., 2011), suggesting that inactivation of RhoA by simvastatin may result in the perturbation of membrane excitation and impairment of calcium influx. RhoA/ROCK pathway also contributes to the sustenance of tonic tension by regulating the phenomenon called Ca2+ sensitization. ROCK inactivates MLC phosphatase through the phosphorylation of MYPT1 and CPI-17, which increases the sensitivity of contractile machinery to Ca2+ and generates greater forces of tension at a certain level of Ca2+ . Ca2+ sensitization and tonic tension play crucial roles in the maintenance of basal vas-

cular tone and regulation of blood flow (de Godoy and Rattan, 2011), suggesting that the impairment of RhoA-regulated functions of vascular smooth muscle by simvastatin may eventually lead to irreversible and adverse effects in vascular integrity. The vascular remodeling may be caused by contractile dysfunction and apoptosis induced by simvastatin. VSMC possesses a high degree of plasticity and easily undergoes phenotypic changes in response to local environmental changes (Fisher, 2010). Contractile function of VSMCs is essential to the maintenance of the contractile phenotype. Contraction of cells stimulates the stretching of cytoskeleton, which transduces the transcriptional signals through FAK, ERK, Src kinases, and integrins that are vital to the maintenance of tissue integrity (Lehoux et al., 2005). Previously, we demonstrated that other compounds like ginsenoside Rg3 and toxic arsenic metabolite, monomethylarsonous acid, could induce the dysfunction of smooth muscle contraction (Bae et al., 2008; Lee et al., 2010; Lim et al., 2011), which eventually resulted in apoptosis and vascular remodeling in vivo. These lines of evidences indicate that the perpetuation of house-keeping functional machinery is essential to the maintenance of structural integrity and the survival, and accordingly the perturbation of the function may ultimately induce permanent damages to structural integrity.

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FIG. 4. Effect of simvastatin on RhoA activation. (A) Schematic of the cholesterol biosynthesis and protein prenylation pathway. After aortic rings without endothelium were simultaneously treated with simvastatin (10␮M) and one of HMG-CoA reductase intermediates, mevalonate (MEV; 100␮M), geranylgeraniol (GGOH; 30␮M), or squalene (SQ; 100mM), for 24 h, (B) PE (10−5 M) or (C) Bay K8644 (10−7 M) induced vessel contraction was measured. (D) Effect of simvastatin on PE (10−5 M) enhanced RhoA activation in primary cultured rat VSMCs was determined by Western blot analysis. Effects of simvastatin on PE (10−5 M) enhanced phosphorylation of (E) MYPT1 and (F) CPI-17 were also observed after simultaneous treatment of simvastatin and/or GGOH for 24 h in aortic rings without endothelium. Values are mean ± SEM of more than four independent experiments. * represents significant differences from the controls (p < 0.05). # represents significant differences from the simvastatin-treated group (p < 0.05).

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FIG. 5. Apoptosis induced by simvastatin in vascular smooth muscle. (A) After aortic rings were treated with 10␮M of simvastatin for 24 h, apoptosis in vascular smooth muscle was evaluated by TUNEL staining (upper panel). TUNEL-positive cells were counted (lower panel). Values are mean ± SEM of more than four independent experiments. * represents significant differences from the controls (p < 0.05). # represents significant differences from the simvastatin-treated group (p < 0.05). (B) Active caspase-3 and procaspase-3 were detected using Western blot analysis after 48 h incubation with simvastatin in primary VSMCs. Representative images were obtained from five independent experiments.

FUNDING

National Research Foundation of Korea, Korea Government (MSIP) (2012R1A2A2A01011705).

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FIG. 6. Effect of simvastatin on PE-induced vasoconstriction ex vivo and vascular remodeling in vivo. Rats were administered with simvastatin 80 mg/kg/day orally and GGOH 10 mg/kg/day intraperitoneally for 7 days. (A) The effects of simvastatin on PE-induced vasoconstriction were determined in intact aortic rings isolated after sacrifice. Values are mean ± SEM of four animals. (B) At the day of sacrifice, the thoracic aorta were fixed in a buffered formalin solution (10%) and embedded in paraffin and horizontal sections were stained with hematoxylin and eosin. Luminal and media area of the thoracic aorta were measured according to the method described. Values are mean ± SEM of eight animals. * represents significant differences from the controls (p < 0.05). # represents significant differences from the simvastatin-treated group (p < 0.05).

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In conclusion, we demonstrated that simvastatin can induce irreversible smooth muscle dysfunction, apoptosis, and vascular remodeling through the disruption of VOCC-mediated intracellular calcium signaling and impairment of RhoA-mediated tonic tension. These effects were reversed by the supplementation of mevalonate and GGOH, suggesting that alteration of protein prenylation and the impairment of RhoA may be at the center of the vascular toxicity of simvastatin. Contractile dysfunction, apoptosis, and structural remodeling in normal vascular smooth muscle may parallel the toxicity of statins in skeletal muscle, suggesting vascular smooth muscle also might be a target of myotoxicity of statins. We believe that this study has enlightened the potential vascular toxicity associated with unnecessarily high and extended use of statins, although future investigation should be done to study its pathological significance.

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