Statins Protect Human Aortic Smooth Muscle Cells From Inorganic Phosphate-Induced Calcification by Restoring Gas6-Axl Survival Pat
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Bo-Kyung Son, Koichi Kozaki, Katsuya Iij
参见附件。
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
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.
Abstract
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
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 stenosis6–8 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
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
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).
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).
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).
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.
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.
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.
Discussion
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 studies6,7,9 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.
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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
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.
Abstract
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
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 stenosis6–8 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
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
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).
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).
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).
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.
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.
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.
Discussion
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 studies6,7,9 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.
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