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(Circulation Research. 2006;98:1024.)
© 2006 American Heart Association, Inc.

Molecular Medicine

Statins Protect Human Aortic Smooth Muscle Cells From Inorganic Phosphate-Induced Calcification by Restoring Gas6-Axl Survival Pathway

Bo-Kyung Son, Koichi Kozaki, Katsuya Iijima, Masato Eto, Taro Kojima, Hidetaka Ota, Yuka Senda, Koji Maemura, Toru Nakano, Masahiro Akishita, Yasuyoshi Ouchi

From the Departments of Geriatric Medicine (B.-K.S., K.K., K.I., M.E., T.K., H.O., Y.S., M.A., Y.O.) and Cardiovascular Medicine (K.M.), Graduate School of Medicine, The University of Tokyo; and Discovery Research Laboratory (T.N.), Shionogi & Co Ltd, Osaka, Japan. Current address for K.K.: Department of Geriatric Medicine, Kyorin University School of Medicine, Tokyo, Japan.

Correspondence to Yasuyoshi Ouchi, MD, PhD, Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail youchi-tky{at}umin.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular calcification is clinically important in the development of cardiovascular disease. It is reported that hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) inhibited vascular calcification in several clinical trials. However, the mechanism is poorly understood. Recently, it has been suggested that apoptosis is one of the important processes regulating vascular smooth muscle cell (VSMC) calcification. In this study, we investigated the effect of statins on VSMC calcification by testing their effect on apoptosis, focusing in particular on regulation of the survival pathway mediated by growth arrest-specific gene 6 (Gas6), a member of the vitamin K–dependent protein family, and its receptor, Axl. In human aortic smooth muscle cells (HASMC), statins significantly inhibited inorganic phosphate (Pi)-induced calcification in a concentration-dependent manner (reduced by 49% at 0.1 µmol/L atorvastatin). The inhibitory effect of statins was mediated by preventing apoptosis, which was increased by Pi in a concentration-dependent manner, and not by inhibiting sodium-dependent phosphate cotransporter (NPC) activity, another mechanism regulating HASMC calcification. Furthermore, the antiapoptotic effect of statins was dependent on restoration of Gas6, whose expression was downregulated by Pi. Restoration of Gas6 mRNA by statins was mediated by mRNA stabilization, and not by an increase in transcriptional activity. Suppression of Gas6 using small interfering RNA and the Axl-extracellular domain abolished the preventive effect of statins on Pi-induced apoptosis and calcification. These data demonstrate that statins protected HASMC from Pi-induced calcification by inhibiting apoptosis via restoration of the Gas6-Axl pathway.

Key Words: calcification • statins • apoptosis • Gas6 • Axl

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular calcification, such as coronary and aortic calcification, is a significant feature of vascular pathology, because this lesion is associated with cardiovascular disease.^1,2 It has been recognized that statins exhibit various protective effects against atherosclerosis, including modification of endothelial function,³ decreased inflammation,⁴ and inhibition of vascular smooth muscle cell (VSMC) proliferation and migration,⁵ all of which cannot be accounted for by lipid reduction. One of the interesting pleiotropic effects of statins is the inhibition of vascular calcification. Results from clinical trials suggest an association of statin use with slowed progression of calcific aortic stenosis^6–8 and coronary artery calcification.⁹ Statins also inhibited calcification of atherosclerotic plaques in experimental hyperlipidemic animals.^10,11 On the other hand, some recent clinical trials were not able to find such an inhibitory effect.^12,13 To clarify these discrepancies, it is important to identify the detailed regulatory mechanism of vascular calcification and the target of effect of statins.

Based on clinical findings,¹⁴ inorganic phosphate (Pi) has been shown to be an important inducer of VSMC calcification, which is morphologically similar to that observed in calcified human heart valves and the aortic media. Transport of Pi into VSMC has been suggested to play an important role in the initiation of extracellular matrix calcification.¹⁵ Recently, it has been shown that similar structures to matrix vesicles, derived from apoptotic VSMC, have been identified in human calcified arteries.¹⁶ These vesicles have the capacity to concentrate and crystallize Ca, initiating calcification. Pi has been shown to induce apoptosis of hypertrophic chondrocytes, which is associated with cell maturation and extracellular matrix mineralization.¹⁷ However, it is not clear whether or not apoptosis plays a regulatory role in the occurrence of VSMC calcification induced by Pi.

Recently, it was shown that growth arrest-specific gene 6 (Gas6), a member of the vitamin K–dependent protein family, and its receptor, Axl, a membrane receptor tyrosine kinase, are decreased on calcification of vascular pericytes.¹⁸ Gas6 is a secreted protein that harbors a -carboxylglutamic acid–rich domain and 4 epidermal growth factor–like repeats.¹⁹ Gas6-Axl interaction has been shown to be implicated in the regulation of multiple cellular functions, including growth, survival, adhesion, and chemotaxis.^20–23 In particular, they are known to protect a range of cell types from apoptotic death. However, there is no evidence that Gas6-Axl interaction is involved in Pi-induced apoptosis and calcification of VSMC.

In the present study, we found that statins inhibited Pi-induced calcification by preventing apoptosis in human aortic smooth muscle cells (HASMC). The effect of statins was dependent on restoration of the Gas6-Axl pathway. Furthermore, this beneficial effect was mediated by Gas6 mRNA stabilization, and not by increasing the transcription rate. Our results reveal a novel pathway by which statins regulate Pi-induced calcification in HASMC.

	Materials and Methods

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Materials
Pravastatin, atorvastatin, and fluvastatin were supplied by Sankyo Co Ltd, Pfizer Inc (New York), and Tanabe Seiyaku Co Ltd, respectively. Recombinant human Gas6 (rhGas6) and Axl-ECD were prepared as described previously.^22,24 All other reagents were of analytical grade.

Cell Culture
HASMC were obtained from Clonetics. They were cultured in DMEM supplemented with 20% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in a humidified atmosphere with 5% CO₂. HASMC were used up to passage 8 for the experiments.

Induction and Quantification of Calcification
For Pi-induced calcification, Pi (a mixed solution of Na₂HPO₄ and NaH₂PO₄ whose pH was adjusted to 7.4) was added to serum-supplemented DMEM to final concentrations of 2.0, 2.6, and 3.2 mmol/L ("calcification medium"). After the indicated incubation period, cells were decalcified with 0.6 mol/L HCl, and Ca content in the supernatant was determined by the o-cresolphthalein complexone method (C-Test, WAKO). The remaining cells were solubilized in 0.1 mol/L NaOH/0.1% SDS, and cell protein content was measured by Bio-Rad protein assay. Calcification was visualized by von Kossa’s method. Briefly, the cells were fixed with 4% formaldehyde and exposed to 5% aqueous AgNO₃.

Induction of Apoptosis
Two different time courses were tested to investigate Pi-induced apoptosis and examine the effect of statins. (1) Short-term condition: Pi was added at final concentrations of 2.0, 2.6, and 3.2 mmol/L for 24 hours at confluence, after the cells were incubated with serum-free DMEM for 48 hours. To test the effect of statins on apoptosis, they were added 24 hours after incubating the cells with serum-free DMEM (12 hours before adding Pi). (2) Long-term condition: at confluence, the medium was switched to calcification medium and cells were cultured for up to 10 days. The medium was changed every 2 days. To test the effect of statins, each was added simultaneously when the medium was switched to the calcification medium.

RNA Extraction, Northern Blot, and mRNA Stability Analysis
The 304-bp product of the Gas6 cDNA probe (forward, 5'-GCGTGGCCAAGAGTGTGAAGT-3'; reverse, 5'-CGCCACTCCTCAACAGAGAT-3') was amplified by RT-PCR. For Northern blot analysis, harvested RNA (5 to 10 µg) was fractionated on 1.4% formaldehyde-agarose gel and transferred to a nylon filter. The filter was hybridized at 68°C for 2 hours with ³²P-labeled Gas6 cDNA and 18S probe in QuickHyb solution (Stratagene) and autoradiographed. To examine Gas6 mRNA stability, serum-starved HASMC were incubated with actinomycin D (Act D, 5 µg/mL) in the presence of 2.6 mmol/L Pi after 12 hours of atorvastatin (0.1 µmol/L) treatment. Total RNA was harvested at 0, 1, 3, and 6 hours for Northern blot analysis. Signal density of the Gas6 mRNA was normalized to that of the 18S RNA at each time point, and the half-life was calculated by linear extrapolation.

Preparation of Small Interfering RNA Targeting Gas6 and Transfection
Two small interfering RNAs (siRNAs) were designed to target human Gas6 (accession no. NM_000820) using siRNA design software (Dharmacon). The sequences for Gas6 were 5'-GGACCTGCCAAGACATAGA-3' and 5'-ACCTCGTGCAGCCTATAAA-3'. Nonspecific control siRNA was synthesized using standard templates (Dharmacon). Twenty-four hours after HASMC seeding onto 12-well plates, cells were cultured in serum-free medium for an additional 24 hours, then transfected with Gas6 (100 nmol/L) and control siRNA using transfection reagent (Upstate). To evaluate the effect of Gas6 siRNA on Ca deposition, siRNA was transfected when HASMC had reached 80% to 90% confluence and then transfected every time the medium was changed (every 2 days) up to 6 days. The loss of Gas6 by transfection of siRNA was validated by immunoblotting for Gas6 protein in the cell lysates 48 hours and 6 days after siRNA transfection.

Statistical Analysis
All results are presented as mean±SEM. Statistical comparisons were made by ANOVA, unless otherwise stated. A value of P<0.05 was considered to be significant.

An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.

	Results

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Statins Inhibit Pi-Induced HASMC Calcification
To induce HASMC calcification, cells were incubated with calcification medium for 10 days. We confirmed that high phosphate (2.6 mmol/L) induced Ca deposition in a concentration- and time-dependent manner, whereas 1.4 mmol/L Pi, equivalent to the human physiological serum phosphate level, was not able to induce Ca deposition up to 10 days. To investigate the effect of statins on Pi-induced calcification, HASMC were incubated with atorvastatin in the presence of 2.6 mmol/L Pi. On day 6, Ca deposition was significantly suppressed by atorvastatin in a concentration-dependent manner (51.1±1.9% of control at 0.1 µmol/L) (Figure 1A). An inhibitory effect of the statins on Ca deposition was also found by von Kossa’s staining (Figure 1B). Atorvastatin was able to be added at as high a concentration as 0.1 µmol/L without cell damage. The inhibitory effect was also observed with fluvastatin (0.001 to 0.1 µmol/L) and pravastatin (0.01 to 50 µmol/L) (data not shown). The inhibitory effect of statins was not blocked by mevalonate (100 µmol/L), farnesylpyrophosphate (1 µmol/L),or geranylgeranylpyrophosphate (1 µmol/L), suggesting that the effect is not dependent on the mevalonate pathway (Figure 1C).

View larger version (46K): [in this window] [in a new window]   Figure 1. Statins prevent HASMC calcification. A, HASMC were cultured with the indicated concentrations of atorvastatin in the presence of 2.6 mmol/L Pi for 6 days. Ca deposition was measured by o-cresolphthalein complexone method and normalized by cell protein content. All values are presented as mean±SEM (n=6). *P<0.05 vs statin (-) by Fisher’s test. N.D. indicates not detected. B, On day 6, the inhibitory effect of atorvastatin (0.1 µmol/L) on 2.6 mmol/L Pi-induced Ca deposition was evaluated at the light microscopic level with von Kossa’s staining. The arrow points to an area of Ca deposition. C, HASMC were cultured with mevalonate (100 µmol/L), farnesylpyrophosphate (1 µmol/L), or geranylgeranylpyrophosphate (1 µmol/L) in the presence of atorvastatin (0.1 µmol/L) and 2.6 mmol/L Pi for 6 days. All values are presented as mean±SEM (n=6).

Inhibitory Effect of Statins on Calcification Is Caused by Preventing Apoptosis, Not by Inhibiting Sodium-Dependent Phosphate Cotransporter Activity
Two different time courses were tested to examine the effect of Pi on HASMC apoptosis: short-term (up to 24 hours) and long-term (up to 10 days; practical time course of calcification process). During calcification, Pi increased the rate of apoptotic cell death detected by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay (Figure 2A). Furthermore, cytoplasmic histone-associated DNA fragments determined by ELISA, as a quantitative index of apoptosis, were also increased by Pi in a concentration- and time-dependent manner in both short-term (Figure 2B) and long-term conditions (supplemental Figure I). In addition, caspase 3 activation, detected by immunoblotting, by 2.6 mmol/L Pi was observed in short-term and long-term conditions (data not shown). To investigate the relationship between apoptosis and calcification, we used ZVAD.fmk, a general caspase inhibitor. We found that ZVAD.fmk significantly inhibited Pi-induced apoptosis as well as calcification in a concentration-dependent manner (Figure 2C).

View larger version (26K): [in this window] [in a new window]   Figure 2. Pi induces apoptosis, and ZVAD.fmk inhibits Pi-induced calcification. A, After incubation with 1.4 (Pi-) and 3.2 mmol/L (Pi+) Pi for 10 days, apoptotic cells were identified by TUNEL staining (green). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). B, Serum-starved HASMC were cultured with the indicated concentration of Pi for 24 hours. A quantitative index of apoptosis, determined by ELISA, is presented as the relative value to that with 1.4 mmol/L Pi. All values are presented as mean±SEM (n=3). *P<0.05 vs 1.4 mmol/L Pi by Fisher’s test. C, HASMC were incubated with the indicated concentration of ZVAD.fmk in the presence of 2.6 mmol/L Pi for 6 days. Ca content was measured and normalized by cell protein content. All values are presented as mean±SEM (n=6). **P<0.01 vs 2.6 mmol/L Pi, ZVAD.fmk(-) by Fisher’s test. Experiments were performed with at least 3 different cell populations.

It has been reported that sodium-dependent phosphate cotransporter (NPC) activity is an important pathway regulating Pi-induced HASMC calcification.²⁵ We confirmed that type III NPC (Pit-1) was expressed in the HASMC that we used, and its activity was enhanced by Pi treatment. Furthermore, a specific inhibitor of NPC, phosphonoformic acid (PFA), inhibited Ca deposition (reduced by 90.4% at 0.1 µmol/L), indicating that NPC-mediated Pi uptake is also essential for HASMC calcification.

To investigate the mechanisms of these statins, we examined the effect of atorvastatin on apoptosis and NPC activity. Atorvastatin, at concentrations exerting inhibition of calcification, reduced apoptosis in a concentration-dependent manner (Figure 3A). A beneficial effect of statins was also observed in the long-term condition (supplemental Figure II). On the other hand, statins did not inhibit NPC activity induced by Pi treatment (Figure 3B).

View larger version (19K): [in this window] [in a new window]   Figure 3. Effect of atorvastatin on Pi-induced apoptosis and NPC activity. A, HASMC were cultured with the indicated concentration of atorvastatin for 12 hours and then incubated with 2.6 mmol/L Pi for an additional 24 hours. All values are presented as mean±SEM (n=3). *P<0.05 vs 2.6 mmol/L Pi, statin (-) by Fisher’s test. B, HASMC were treated with (dotted line) or without (solid line) 0.1 µmol/L atorvastatin in the presence of 2.6 mmol/L Pi. On day 6, NPC activity was determined in Earl’s balanced salt solution containing 0.1 mmol/L H332PO4 (1 µCi/mL) with 143 mmol/L sodium chloride for the indicated period. All values are presented as mean±SEM (n=6).

Downregulation of Gas6-Axl Interaction Is Associated With Pi-Induced Apoptosis
Immunoblot analysis showed that the expression of Gas6 and Axl was markedly downregulated by 2.6 mmol/L Pi in both short-term (Figure 4A) and long-term (supplemental Figure III) conditions. To further examine whether Pi affects the secretion of Gas6 by HASMC, conditioned medium was collected after Pi treatment. Gas6 production in the medium was reduced by 2.6 mmol/L Pi, along with a reduction in its intracellular expression (Figure 4B). Gas6 production was also reduced in an immunoprecipitation-immunoblotting study on day 10 (Figure 4C). Next, to investigate the role of Gas6-Axl interaction in the process of apoptosis and calcification, rhGas6 and Axl-ECD were supplemented in Pi-treated HASMC. The addition of rhGas6 significantly inhibited both Pi-induced apoptosis and calcification. Addition of Axl-ECD to block the binding of Gas6 to Axl clearly abrogated the inhibitory effect of rhGas6 (Figure 4D and 4E). These results indicate that Pi-induced apoptosis and calcification are associated with downregulation of the Gas6-Axl interaction.

View larger version (47K): [in this window] [in a new window]   Figure 4. Pi reduces production of Gas6 and Axl, and rhGas6 inhibits Pi-induced apoptosis and calcification via Axl. A, HASMC were cultured in the presence of 2.6 mmol/L Pi for 12 hours. Cell lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies to Gas6, Axl, or ß-tubulin. B, Conditioned medium of HASMC in the absence (lane 1) or presence (lane 2) of 2.6 mmol/L Pi at 12 hours was concentrated and separated by SDS-PAGE along with cell lysates. C, Conditioned medium of HASMC on day 10 in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 2.6 mmol/L Pi was subjected to immunoprecipitation with anti-Gas6 antibody (lanes 1 and 2) or control goat IgG (lanes 3 and 4). Precipitates were immunoblotted with anti-Gas6 antibody. D, After pretreatment with rhGas6 (400 ng/mL) with or without Axl-ECD (1 µg/mL), apoptosis was induced by 2.6 mmol/L Pi. All values are presented as mean±SEM (n=3). *P<0.05 by Fisher’s test. E, For measurement of Ca deposition, HASMC were cultured with rhGas6 (400 ng/mL) with or without Axl-ECD (1 µg/mL) in the presence of 2.6 mmol/L Pi for 6 days. All values are presented as mean±SEM (n=6). *P<0.05 by Fisher’s test. Experiments were performed with at least 3 different cell populations.

Statin-Mediated Induction of Gas6 Expression Is Dependent on mRNA Stabilization, Not on Transcription
To investigate whether the antiapoptotic effect of statins is dependent on restoration of the Gas6-Axl interaction, we first assessed the effect of statins on Gas6 expression. As shown in Figure 5A, atorvastatin increased Gas6 expression, which was downregulated by Pi at both the mRNA and protein levels. Upregulation of Gas6 expression was also observed in the long-term condition (supplemental Figure IV). Furthermore, to elucidate the mechanism of statins on restoration of Gas6 mRNA, a promoter study was undertaken. Reporter assay using the –1.9 kb Gas6-luciferase DNA construct revealed that atorvastatin did not have a significant effect on Gas6 promoter activity (supplemental Figure V), as well as mRNA expression under the condition in which it was significantly inhibited by PDGF-BB (data not shown). Next, we investigated the effect of atorvastatin on mRNA stabilization using an RNA polymerase inhibitor, actinomycin D (ActD). As shown in Figure 5B, Gas6 mRNA expression was more stable in the presence of atorvastatin than in its absence under Pi and ActD treatment. The half-life was 15.9 hours with atorvastatin and 5 hours without atorvastatin, suggesting the capacity of statins to improve Gas6 mRNA stabilization (Figure 5C). Taken together, these findings suggest that the restoration of Gas6 mRNA by statins appears to be mediated by decreasing the mRNA degradation rate, and not by stimulating transcriptional activity.

View larger version (32K): [in this window] [in a new window]   Figure 5. Atorvastatin enhances Gas6 mRNA stabilization, but not transcription. A, After pretreatment with atorvastatin (0.1 µmol/L) for 12 hours, apoptosis was induced by 2.6 mmol/L Pi. At 12 hours, mRNA was isolated and Northern blot analysis for Gas6 and 18S was performed. Simultaneously, cell lysates were collected and subjected to SDS-PAGE followed by immunoblotting with antibodies to Gas6 and ß-tubulin. B, Serum-starved HASMC were incubated with actinomycin D (Act D) (5 µg/mL) in the presence of 2.6 mmol/L Pi after 12 hours of atorvastatin (0.1 µmol/L) treatment. Total RNA was harvested at 0, 1, 3, and 6 hours for Northern blot analysis. C, Signal density of Gas6 mRNA with (solid line) or without (dotted line) atorvastatin (0.1 µmol/L) in the presence of 2.6 mmol/L Pi and Act D (5 µg/mL) was normalized to that of 18S RNA at each time point. Gas6 mRNA level at time 0 was given the value 1. Each experiment was performed in triplicate for each condition.

Furthermore, to determine whether Gas6 is required for statin-mediated effects, we tried to knock down the action of Gas6 and examined the effect of atorvastatin on Pi-induced apoptosis and calcification. Transfection of Gas6 siRNA markedly decreased Gas6 expression in the short-term and long-term conditions (Figure 6A). The inhibitory effect of atorvastatin on Pi-induced apoptosis and calcification was reversed by Gas6 siRNA (Figure 6B and 6C). Similarly, the beneficial effect of atorvastatin was also abolished by blocking the binding of Gas6 to Axl using Axl-ECD (Figure 6D and 6E). These data support a critical role of Gas6 in the preventive effect of statins on apoptosis and calcification.

View larger version (37K): [in this window] [in a new window]   Figure 6. Gas6 knockdown abolishes inhibition of Pi-induced apoptosis and calcification by atorvastatin. A, Gas6-specific siRNA (100 nmol/L) and nonspecific siRNA (Ctrl siRNA) were transfected into HASMC, and immunoblotting was performed at 48 hours and 6 days after transfection. B, Serum-starved HASMC were transfected with 100 nmol/L Gas6 siRNA and control (Ctrl) siRNA. After transfection, cells were treated with atorvastatin (0.1 µmol/L) for 12 hours, then with 2.6 mmol/L Pi for an additional 24 hours before measurement of apoptosis (n=3). C, For measurement of Ca deposition, HASMC were transfected with 100 nmol/L Gas6 siRNA and control siRNA and incubated with atorvastatin (0.1 µmol/L) and 2.6 mmol/L Pi for 6 days (n=3). D, In the case of Axl-ECD, HASMC were pretreated with atorvastatin (0.1 µmol/L) and Axl-ECD (1 µg/mL) for 12 hours, then incubated with 2.6 mmol/L Pi for an additional 24 hours. Thereafter, a quantitative index of apoptosis was determined by ELISA (n=3). E, HASMC were cultured with atorvastatin (0.1 µmol/L) and Axl-ECD (1 µg/mL) in the presence of 2.6 mmol/L Pi for 6 days. Ca content was measured and normalized by cell protein content. All values are presented as mean±SEM (n=6). *P<0.05 by Fisher’s test. Each panel shows a representative example of 3 independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrated that statins protected HASMC from Pi-induced calcification. The clinical effect of statins on vascular calcification is controversial. Many retrospective clinical studies^6,7,9 and a prospective study⁸ have shown beneficial effects, whereas recent prospective studies were unable to show such effects.^12,13 The reason is not yet clear, and the time window of statin use has been raised as an important matter. The discrepancy may also derive from the complex in vivo effects of statins. In this regard, it is important to analyze the detailed regulatory mechanism of statins in a simple model.

In Pi-induced calcification, HASMC undergo apoptosis. A causal link between apoptosis and calcification was evident from the finding that both apoptosis and calcification were inhibited by the general caspase inhibitor, ZVAD.fmk. As reported previously,²⁵ we confirmed that NPC-mediated Pi uptake is another essential mechanism for HASMC calcification. Given that apoptosis does not always lead to calcification, Pi-induced HASMC calcification is presumably dependent on both an NPC-mediated phenotypic transition from SMC to an osteoblastic phenotype and apoptotic cell death. With respect to the mechanism of action of statins, they clearly inhibited Pi-induced apoptosis, although they did not have an effect on Pi-induced NPC activity or osteoblastic differentiation; Pi-induced upregulation of matrix Gla protein (MGP) mRNA was not inhibited by atorvastatin (supplemental Figure VI). These results suggest that apoptosis is the target of statins in inhibiting HASMC calcification.

Another important signal in Pi-induced calcification is an increase in intracellular Ca ([Ca²⁺]_i). Statins have been shown to inhibit VSMC proliferation⁵ and reduce the acute increase of [Ca²⁺]_i in a mevalonate and isoprenoid pathway–independent manner.²⁶ On the other hand, [Ca²⁺]_i is reported to modulate Pi-induced apoptosis of terminally differentiated chondrocytes.²⁷ Therefore, modulation of [Ca²⁺]_i is another possible mechanism of the inhibition of apoptosis by statins. In this study, we investigated the association of proliferation with Pi-induced apoptosis and calcification. We found that Pi did not affect proliferation, measured by the incorporation of 5-bromo-2'-deoxyuridine (BrdU) during calcification (data not shown). We also found that the inhibitory effect of statins on calcification was not affected by an inhibitor of Rho kinase (Y-27632), an important modulator of the mevalonate and isoprenoid pathway affecting proliferation and apoptosis (supplemental Figure VII). These results suggest that proliferation is not associated with Pi-induced calcification. The inhibitory effect of statins on calcification was not blocked by mevalonate, farnesylpyrophosphate, geranylgeranylpyrophosphate, or Rho kinase inhibitor, suggesting that the effect of statins is not dependent on the mevalonate and isoprenoid pathways. Indeed, a mevalonate pathway–independent effect of statins has been reported previously,^26,28–30 although the precise mechanism has not been shown. The pleiotropism of statins is of continuing interest.

An antiapoptotic effect of statins has been shown in various cell types.^31–34 In cardiomyocytes, apoptosis induced by hypoxia or protein kinase C (PKC) inhibitors was inhibited by 10 µmol/L pravastatin or 0.1 µg/mL atorvastatin, respectively.^31,32 Simvastatin (1 µmol/L) promoted endothelial cell survival.³³ In VSMC, 7-ketocholesterol–induced apoptosis was inhibited by 10 µmol/L pravastatin.³⁴ However, in contrast to the results of the present and other studies, a proapoptotic effect of statins has also been reported in VSMC,³⁵ endothelial cells,³⁶ and cardiac myocytes.³⁷ Although the precise mechanism is not understood, it can be postulated that statins have biphasic effects on cell survival (an antiapoptotic effect at low concentrations and a proapoptotic effect at high concentrations) depending on the type of cell, statins, and apoptotic stimulus. Indeed, Weis et al showed dose-dependent biphasic effects of statins on apoptotic activity in microvascular endothelial cells.³⁰ Consistent with these data, we found that 3 different statins displayed an antiapoptotic effect at low concentrations and a proapoptotic effect at high concentrations (>1 µmol/L for atorvastatin and fluvastatin; >100 µmol/L for pravastatin) (data not shown).

During Pi-induced apoptosis, we have shown that Pi downregulates the Gas6-Axl interaction, resulting in blockade of a survival signal, thereby promoting apoptosis and calcification. We previously proposed that Gas6 may allow Axl-expressing phagocytic cells, eg, macrophages and VSMC, to recognize cells exposing phosphatidylserine (PS) on the outer cell membrane, the initial step of the apoptotic process.³⁸ Proudfoot et al also showed that in vascular calcification, several PS-exposing cells are observed within and on the periphery of the nodules.¹⁶ PS exposure by apoptotic bodies generates a potential Ca-binding site and membrane surface suitable for hydroxyapatite deposition.^39,40 Based on these observations, Gas6-Axl downregulation is presumably involved in decreased cell survival and clearance, both directing cells to apoptosis-mediated mineral deposition.

With regard to the molecular pathway of the restoration of Gas6 by statins, we have shown that statins retarded degradation of Gas6 mRNA, not increasing the transcriptional rate. Indeed, it was reported that statins improve mRNA stability as well as transcription.^41,42 In addition, the result that suppression of the action of Gas6 by siRNA and Axl-ECD abrogated the inhibitory effect of statins on apoptosis and inhibition clearly indicates a pivotal role of Gas6 in the effect of statins.

We conclude that statins inhibit Pi-induced HASMC calcification by preventing apoptosis via restoration of the Gas6-Axl pathway. The regulation of Gas6 by statins occurs at the posttranscriptional level. The present study provides evidence of a preventive role of statins in vascular calcification and further indicates the pleiotropic effects of statins, which could potentially contribute to the treatment of cardiovascular disease.

	Acknowledgments

 
This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan (grant 15390239) and by the Mitsui Sumitomo Insurance Welfare Foundation, the Ono Medical Research Foundation, the Kanzawa Medical Research Foundation, the Novartis Foundation for Gerontrogical Research, and the Takeda Research Foundation. We thank Yuki Ito for technical assistance.

	Footnotes

 
Original received April 19, 2005; revision received August 8, 2005; resubmission received February 20, 2006; accepted March 14, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Eggen DA. Relationship of calcified lesions to clinically significant atherosclerotic lesions. Ann N Y Acad Sci. 1968; 149: 752–767.[Medline] [Order article via Infotrieve]
2. Wexler L, Brundage B, Crouse J, Detrano R, Fuster V, Maddahi J, Rumberger J, Stanford W, White R, Taubert K. Coronary artery calcification: pathophysiology, epidemiology, imaging methods, and clinical implications. A statement for health professionals from the American Heart Association Writing Group. Circulation. 1996; 94: 1175–1192.[Free Full Text]
3. Mullen MJ, Wright D, Donald AE, Thorne S, Thomson H, Deanfield JE. Atorvastatin but not L-arginine improves endothelial function in type-I diabetes mellitus: a double-blind study. J Am Coll Cardiol. 2000; 36: 410–416.[Abstract/Free Full Text]
4. Bustos C, Hernandez-Presa MA, Ortego M, Tunon J, Ortega L, Perez F, Diaz C, Hernandez G, Egido J. HMG-CoA reductase inhibition by atorvastatin reduces neointimal inflammation in a rabbit model of atherosclerosis. J Am Coll Cardiol. 1998; 32: 2057–2064.[Abstract/Free Full Text]
5. Axel DI, Riessen R, Runge H, Viebahn R, Karsch KR. Effects of cerivastatin on human arterial smooth muscle cell proliferation and migration in transfilter cocultures. J Cardiovasc Pharmacol. 2000; 35: 619–629.[CrossRef][Medline] [Order article via Infotrieve]
6. Shavelle DM, Takasu J, Budoff MJ, Mao S, Zhao XQ, O’Brien KD. HMG CoA reductase inhibitor (statin) and aortic valve calcium. Lancet. 2002; 359: 1125–1126.[CrossRef][Medline] [Order article via Infotrieve]
7. Novaro GM, Tiong IY, Pearce GL, Lauer MS, Sprecher DL, Griffin BP. Effect of hydroxymethylglutaryl coenzyme A reductase inhibitors on the progression of calcific aortic stenosis. Circulation. 2001; 104: 2205–2209.[Abstract/Free Full Text]
8. Achenbach S, Ropers D, Pohle K, Leber A, Thilo C, Knez A, Menendez T, Maeffert R, Kusus M, Regenfus M, Bickel A, Haberl R, Steinbeck G, Moshage W, Daniel WG. Influence of lipid-lowering therapy on the progression of coronary artery calcification: a prospective evaluation. Circulation. 2002; 106: 1077–1082.[Abstract/Free Full Text]
9. Callister TQ, Raggi P, Cooil B, Lippolis NJ, Russo DJ. Effect of HMG-CoA reductase inhibitors on coronary artery disease as assessed by electron-beam computed tomography. N Engl J Med. 1998; 339: 1972–1978.[Abstract/Free Full Text]
10. Williams JK, Sukhova GK, Herrington DM, Libby P. Pravastatin has cholesterol-lowering independent effects on the artery wall of atherosclerotic monkeys. J Am Coll Cardiol. 1998; 31: 684–691.[Abstract/Free Full Text]
11. Bea F, Blessing E, Bennett B, Levitz M, Wallace EP, Rosenfeld ME. Simvastatin promotes atherosclerotic plaque stability in apoE-deficient mice independently of lipid lowering. Arterioscler Thromb Vasc Biol. 2002; 22: 1832–1837.[Abstract/Free Full Text]
12. Cowell SJ, Newby DE, Prescott RJ, Bloomfield P, Reid J, Northridge DB, Boon NA. A randomized trial of intensive lipid-lowering therapy in calcific aortic stenosis. N Engl J Med. 2005; 352: 2389–2397.[Abstract/Free Full Text]
13. Wanner C, Krane V, Marz W, Olschewski M, Mann JF, Ruf G, Ritz E. Atorvastatin in patients with type 2 diabetes mellitus undergoing hemodialysis. N Engl J Med. 2005; 353: 238–248.[Abstract/Free Full Text]
14. Goodman WG, London G, Amann K, Block GA, Giachelli C, Hruska KA, Ketteler M, Levin A, Massy Z, McCarron DA, Raggi P, Shanahan CM, Yorioka N; Vascular Calcification Work Group. Vascular calcification in chronic kidney disease. Am J Kidney Dis. 2004; 43: 572–579.[CrossRef][Medline] [Order article via Infotrieve]
15. Giachelli CM, Jono S, Shioi A, Nishizawa Y, Mori K, Morii H. Vascular calcification and inorganic phosphate. Am J Kidney Dis. 2001; 38: S34–S37.[Medline] [Order article via Infotrieve]
16. Proudfoot D, Skepper JN, Hegyi L, Bennett MR, Shanahan CM, Weissberg PL. Apoptosis regulates human vascular calcification by apoptotic bodies. Circ Res. 2000; 87: 1055–1062.[Abstract/Free Full Text]
17. Mansfield K, Rajpurohit R, Shapiro IM. Extracellular phosphate ions cause apoptosis of terminally differentiated epiphyseal chondrocytes. J Cell Physiol. 1999; 179: 276–286.[CrossRef][Medline] [Order article via Infotrieve]
18. Collett G, Wood A, Alexander MY, Varnum BC, Boot-Handford RP, Ohanian V, Ohanian J, Fridell YW, Canfield AE. Receptor tyrosine kinase Axl modulates the osteogenic differentiation of pericytes. Circ Res. 2003; 92: 1123–1129.[Abstract/Free Full Text]
19. Mark MR, Chen J, Hammonds RG, Sadick M, Godowsk PJ. Characterization of Gas6, a member of the superfamily of G domain-containing proteins, as a ligand for Rse and Axl. J Biol Chem. 1996; 271: 9785–9789.[Abstract/Free Full Text]
20. Yanagita M, Arai H, Ishii K, Nakano T, Ohashi K, Mizuno K, Varnum B, Fukatsu A, Doi T, Kita T. Gas6 regulates mesangial cell proliferation through Axl in experimental glomerulonephritis. Am J Pathol. 2001; 158: 1423–1432.[Abstract/Free Full Text]
21. Goruppi S, Ruaro E, Schneider C. Gas6, the ligand of Axl tyrosine kinase receptor, has mitogenic and survival activities for serum starved NIH3T3 fibroblasts. Oncogene. 1996; 12: 471–480.[Medline] [Order article via Infotrieve]
22. Nakano T, Ishimoto Y, Kishino J, Umeda M, Inoue K, Nagata K, Ohashi K, Mizuno K, Arita H. Cell adhesion to phosphatidylserine mediated by a product of growth arrest-specific gene 6. J Biol Chem. 1997; 272: 29411–29414.[Abstract/Free Full Text]
23. Fridell YW, Villa J Jr, Attar EC, Liu ET. Gas6 induces Axl-mediated chemotaxis of vascular smooth muscle cells. J Biol Chem. 1998; 273: 7123–7126.[Abstract/Free Full Text]
24. Ming Cao W, Murao K, Imachi H, Sato M, Nakano T, Kodama T, Sasaguri Y, Wong NC, Takahara J, Ishida T. Phosphatidylinositol 3-OH kinase-Akt/protein kinase B pathway mediates Gas6 induction of scavenger receptor a in immortalized human vascular smooth muscle cell line. Arterioscler Thromb Vasc Biol. 2001; 21: 1592–1597.[Abstract/Free Full Text]
25. Jono S. McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, Morii H, Giachelli CM. Phosphate regulation of vascular smooth muscle cell calcification. Circ Res. 2000; 87: e10–e17.[Medline] [Order article via Infotrieve]
26. Bergdahl A, Persson E, Hellstrand P, Sward K. Lovastatin induces relaxation and inhibits L-type Ca(2+) current in the rat basilar artery. Pharmacol Toxicol. 2003; 93: 128–134.[CrossRef][Medline] [Order article via Infotrieve]
27. Mansfield K, Pucci B, Adams CS, Shapiro IM. Induction of apoptosis in skeletal tissues: phosphate-mediated chick chondrocyte apoptosis is calcium dependent. Calcif Tissue Int. 2003; 73: 161–172.[CrossRef][Medline] [Order article via Infotrieve]
28. Weitz-Schmidt G, Welzenbach K, Brinkmann V, Kamata T, Kallen J, Bruns C, Cottens S, Takada Y, Hommel U. Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nat Med. 2001; 7: 687–692.[CrossRef][Medline] [Order article via Infotrieve]
29. Wagner AH, Gebauer M, Guldenzoph B, Hecker M. 3-Hydroxy-3-methylglutaryl coenzyme A reductase-independent inhibition of CD40 expression by atorvastatin in human endothelial cells. Arterioscler Thromb Vasc Biol. 2002; 22: 1784–1789.[Abstract/Free Full Text]
30. Weis M, Heeschen C, Glassford AJ, Cooke JP. Statins have biphasic effects on angiogenesis. Circulation. 2002; 105: 739–745.[Abstract/Free Full Text]
31. Bergmann MW, Rechner C, Freund C, Baurand A, El Jamali A, Dietz R. Statins inhibit reoxygenation-induced cardiomyocyte apoptosis: role for glycogen synthase kinase 3ß and transcription factor ß-catenin. J Mol Cell Cardiol. 2004; 37: 681–690.[CrossRef][Medline] [Order article via Infotrieve]
32. Tanaka K, Honda M, Takabatake T. Anti-apoptotic effect of atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitor, on cardiac myocytes through protein kinase C activation. Clin Exp Pharm Phy. 2004; 31: 360–364.[CrossRef]
33. Kureishi Y, Luo Z, Shiojima I, Bialik A, Fulton D, Lefer DJ. Sessa WC, Walsh K. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nature Med. 2000; 6: 1004–1010.[CrossRef][Medline] [Order article via Infotrieve]
34. Miyashita Y, Ozaki H, Koide N, Otsuka M, Oyama T, Itoh Y, Mastuzaka T, Shirai K. Oxysterol-induced apoptosis of vascular smooth muscle cells is reduced by HMG-CoA reductase inhibitor, pravastatin. J Atheroscler Thromb. 2002; 9: 65–71.[Medline] [Order article via Infotrieve]
35. Guijarro C, Blanco-Colio LM, Ortego M, Alonso C, Ortiz A, Plaza JJ, Diaz C, Hernandez G, Egido J. 3-Hydroxy-3 methylglutaryl coenzyme A reductase and isoprenylation inhibitors induce apoptosis of vascular smooth muscle cells in culture. Circ Res. 1998; 83: 490–500.[Abstract/Free Full Text]
36. Newton CJ, Ran G, Xie YX, Bilko D, Burgoyne CH, Adams I, Abidia A, McCollum PT, Atkin SL. Statin-induced apoptosis of vascular endothelial cells is blocked by dexamethasone. J Endocrinol. 2002; 174: 7–16.[Abstract]
37. Ogata Y, Takahashi M, Takeuchi K, Ueno S, Mano H, Ookawara S, Kobayashi E, Ikeda U, Shimada K. Fluvastatin induces apoptosis in rat neonatal cardiac myocytes: a possible mechanism of statin-attenuated cardiac hypertrophy. J Cardiovasc Pharmacol. 2002; 40: 907–915.[CrossRef][Medline] [Order article via Infotrieve]
38. Ishimoto Y, Ohashi K, Mizuno K, Nakano T. Promotion of the uptake of PS liposomes and apoptotic cells by a product of growth arrest-specific gene, gas6. J Biochem (Tokyo). 2000; 127: 411–417.[Abstract/Free Full Text]
39. Cotmore JM, Nichols G Jr, Wuthier RE. Phospholipid-calcium phosphate complex: enhanced calcium migration in the presence of phosphate. Science. 1971; 172: 1339–1341.[Abstract/Free Full Text]
40. Skrtic D, Eanes ED. Membrane mediated precipitation of calcium phosphate in model liposomes with matrix vesicle-like lipid composition. Bone Miner. 1992; 16: 109–119.[CrossRef][Medline] [Order article via Infotrieve]
41. Walter DH, Zeiher AM, Dimmeler S. Effects of statins on endothelium and their contribution to neovascularization by mobilization of endothelial progenitor cells. Coron Artery Dis. 2004; 15: 235–242.[CrossRef][Medline] [Order article via Infotrieve]
42. Menschikowski M, Hagelgans A, Heyne B, Hempel U, Neumeister V, Goez P, Jaross W, Siegert G. Statins potentiate the IFN-gamma-induced upregulation of group IIA phospholipase A2 in human aortic smooth muscle cells and HepG2 hepatoma cells. Biochim Biophys Acta. 2005; 1733: 157–171.[Medline] [Order article via Infotrieve]

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