Hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) have been shown to reduce cardiovascular mortality and morbidity (1–3). Although the beneficial effects of statins are usually explained in terms of their ability to reduce cholesterol synthesis, increasing evidence suggests that statins also may have direct effects on the vascular wall independent of their hypocholesterolemic properties (4–6). Because mevalonate is a precursor of not only cholesterol, but also a number of nonsteroidal isoprenoid compounds essential for normal cellular activity, the inhibition of hydroxymethylglutaryl coenzyme A reductase may have potential pleiotropic effects. Indeed, experimental models of atherosclerosis demonstrated that statins could attenuate the formation of atherosclerotic lesions and neointimal thickening regardless of lipid reduction (7,8).
Human vascular smooth muscle cells (VSMCs) from atheromatous lesions are more likely to undergo apoptosis than are VSMCs derived from normal arteries (9–11). In addition, apoptosis is more frequent in proliferative lesions, particularly restenotic lesions (12,13), suggesting that apoptosis of VSMCs may play a significant role in the pathophysiology of atherosclerosis and neointimal thickening after vascular injury. In the current study, we investigate the effect of lipophilic and hydrophilic statins (fluvastatin and pravastatin) on apoptosis in unstimulated or cytokine-stimulated VSMCs.
Cell culture and materials
Primary cultures of VSMCs were obtained from the media of thoracic aortas of male Sprague–Dawley rats, as described previously (14). The cells were grown in Dulbecco modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution. The investigation was performed in accordance with the Home Office Guidance on the Operation of Animals (Scientific Procedures) Act, 1986 (Her Majesty's Stationary Office, London, U.K.).
Tanabe Pharmaceutical Co. (Osaka, Japan) and Sankyo Pharmaceutical Co. (Tokyo, Japan), respectively, kindly provided fluvastatin and pravastatin. Stock solutions of fluvastatin and pravastatin were made in water. Human interleukin-1β was a gift from Otsuka Pharmacy (Tokushima, Japan). PD098059 and SB203580 were purchased from Calbiochem (San Diego, CA, U.S.A.). YVAD-FMK and DEVD-FMK were purchased from MBL (Nagoya, Japan). Anti-phospho-extracellular signal-regulated protein kinase (ERK) 1/2 and phospho-p38-mitogen-activated protein kinase (MAPK) polyclonal antibody were obtained from New England Biolabs Inc. (Beverly, MA, U.S.A.). Anti-ERK1/2 and p38-MAPK polyclonal antibody were obtained from Santa-Cruz Biotechnology Inc. (Santa Cruz, CA, U.S.A.). The remaining reagents were obtained from Sigma (St. Louis, MO, U.S.A.) unless specified otherwise.
DNA electrophoresis (DNA laddering)
For the evaluation of DNA fragmentation, cellular fragmented DNA was extracted by the Triton X-100 (Pharmacia Biotech, Uppsala, Sweden) lysis method, which efficiently eliminates intact chromatin. Floating or adherent cells were collected and DNA fragments were extracted, fractionated by 2% agarose gel electrophoresis, and stained with ethidium bromide.
Cells were fixed in 3% paraformaldehyde in phosphate-buffered saline for 20 min and stained with a solution of 4´, 6-diamidino-2-phenylindole (DAPI) (10 m M tromethamine HCl pH 7.4, 10 m M ethylenediamine tetra-acetic acid (EDTA), 100 m M NaCl, 500 ng/ml of DAPI) for 10 min at room temperature. The apoptotic cells were evaluated under a fluorescent microscope (15).
Flow cytometry analysis
Cellular DNA content was assessed by flow cytometry as described previously (16). Briefly, floating or adherent cells were collected, spun and resuspended in 70% ice-cold ethanol, and fixed at 4°C for 4 h. Cells were resuspended in a solution containing 75 μM propidium iodide and 0.05 mg/ml of RNase A, then incubated at room temperature for 30 min in the dark and analyzed by a Becton-Dickinson flow cytometer (Bedford, MA, U.S.A.). The percentage of apoptotic cells with decreased DNA staining, resulting from either DNA fragmentation or condensed chromatin, was determined from counts of a minimum 10,000 cells per experimental condition.
Western blot analysis
Western blotting for phosphorylation and protein levels of ERK1/2 and p38-MAPK was performed as described previously (17). Briefly, cells were washed with ice-cold phosphate-buffered saline and lysed in modified cell lysis buffer (10 m M N-[2-hydroxyethyl]piperazine-N ´-[2-ethanesulfonic acid), pH 7.4, 5 m M EDTA, 5 m M egtazic acid, 50 m M sodium pyrophosphate, 50 m M NaF, 50 m M NaCl, 100 μM Na 3 VO 4, 0.1% Triton X-100, fresh 0.1 m M phenylmethyl sulfonyl fluoride, and 10 μg/ml of leupeptin). Cell lysates were prepared by scraping, sonication, and centrifugation. Sample protein concentrations were determined by DC protein (Bio-Rad, Hercules, CA, U.S.A.) assay. Cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions, and proteins were then transferred to a nitrocellulose membrane. The membrane was blocked for 2 h at room temperature with 5% skim milk, and then incubated for 1 h at room temperature with the primary antibodies, followed by incubation for 1–2 h with secondary antibody (horseradish peroxidase conjugated). Immunoreactive bands were visualized by enhanced chemiluminescence system (Amersham Life Science, Buckinghamshire, UK).
Data are expressed as mean ± SEM. For comparisons between multiple groups, we determined the significance of differences between group means by analysis of variance using the least significant difference for multiple comparisons. Differences at values of p < 0.05 were considered to be statistically significant.
Fluvastatin enhances apoptosis in interleukin-1β-stimulated vascular smooth muscle cells
Electrophoresis of DNA fragments showed that treatment with fluvastatin (0.3–10 μM) for 24 h induced progressive formation of the characteristic apoptosis ladder in interleukin-1β-stimulated VSMCs in a dose-dependent manner, whereas pravastatin had no effect (Fig. 1A). Treatment with fluvastatin, pravastatin, or interleukin-1β (5 ng/ml) alone for 24 h showed no effect on VSMC apoptosis. After 48 h of culture, fluvastatin markedly increased apoptosis in interleukin-1β-stimulated VSMCs, although fluvastatin alone showed no effect. Interleukin-1β alone slightly induced apoptosis. In addition, high concentrations of fluvastatin (30–100 μM) alone induced apoptosis in unstimulated VSMCs, whereas high concentrations of pravastatin (30–100 μM) did not (data not shown). These results indicate that fluvastatin enhanced apoptosis in interleukin-1β-stimulated VSMCs. We further confirmed the fluvastatin-enhanced apoptosis in interleukin-1β-stimulated VSMCs by nuclear morphology using DAPI staining (Fig. 1B) and by flow cytometry analysis (Fig. 1C). These findings indicate that fluvastatin (but not pravastatin) markedly enhances apoptosis in interleukin-1β-stimulated VSMCs, suggesting that the sensitivity of VSMCs on apoptosis to fluvastatin is increased by the treatment with interleukin-1β.
Effects of isoprenoids on fluvastatin-enhanced apoptosis
To evaluate whether mevalonate metabolites can reverse the proapoptotic effect of fluvastatin in interleukin-1β-stimulated VSMCs, the cells were treated with interleukin-1β and fluvastatin in the presence of mevalonate, farnesyl-pyrophosphate (FPP), geranylgeranyl-pyrophosphate (GGPP), or squalene. Squalene is a cholesterol precursor, and FPP and GGPP are involved in farnesylation and geranylgeranylation of proteins, respectively (18). As shown in Figure 2, mevalonate or GGPP completely and FPP partially reversed the effect of fluvastatin, whereas squalene had no effect. Treatment with mevalonate, FPP, GGPP, or squalene alone showed no effect on apoptosis in unstimulated VSMCs (data not shown). These results indicate that the proapoptotic effect of fluvastatin is independent of its lipid-lowering properties and related to changes in protein geranylgeranylation.
Involvement of mitogen-activated protein kinase activation in apoptosis
Recent evidence suggests that MAPK is involved in apoptotic cell death in certain types of cells (19). We therefore hypothesized that MAPK may participate in fluvastatin-enhanced apoptosis in interleukin-1β-stimulated VSMCs. As shown in Figure 3A and B, pretreatment with PD098059 (an ERK1/2 inhibitor) significantly enhanced the fluvastatin-enhanced apoptosis in interleukin-1β-stimulated VSMCs, whereas pretreatment with SB203580 (a p38-MAPK inhibitor) significantly prevented the apoptosis. Interestingly, these two reagents showed no effects on apoptosis in unstimulated or interleukin-1β-stimulated VSMCs. We next examined the activity of ERK1/2 and p38-MAPK in VSMCs by Western blot analysis. As shown in Figure 4, interleukin-1β activated both ERK1/2 and p38-MAPK, whereas fluvastatin did not. In addition, fluvastatin had no additive effects on interleukin-1β-activated ERK1/2 and p38-MAPK. The activation of ERK1/2 and p38-MAPK by interleukin-1β was completely blocked by PD098059 and SB203580, respectively. There were no significant changes in protein levels of ERK1/2 and p38-MAPK during these experiments.
Involvement of caspase inhibition in apoptosis
To further investigate the mechanisms of fluvastatin-enhanced apoptosis in interleukin-1β-stimulated VSMCs, we examined the effects of YVAD-FMK, which is an inhibitor of caspase-1 (interleukin-1β- convertingbgr;-converting enzyme protease), and DEVD-FMK, which is an inhibitor of caspase-3 (CPP32); both enzymes participate in an important common apoptosis signal transduction pathway (20). Both YVAD-FMK and DEVD-FMK significantly inhibited fluvastatin-induced apoptosis in interleukin-1β-stimulated VSMCs (Fig. 5).
The major findings of this study are as follows. The lipophilic statin drug fluvastatin (but not the hydrophilic statin pravastatin) enhanced apoptosis in interleukin-1β-stimulated VSMCs. Both mevalonate, GGPP, and FPP, but not squalene, prevented fluvastatin-enhanced apoptosis. Inhibition of ERK1/2 further enhanced the apoptosis whereas inhibition of p38-MAPK prevented the apoptosis, and inhibition of the caspase pathway inhibited the apoptosis. These findings suggest that fluvastatin enhances apoptosis in interleukin-1β-stimulated VSMCs and that protein prenylation, MAPK (ERK1/2 and p38-MAPK), and caspases are involved in the pathways of fluvastatin-enhanced apoptosis.
Apoptosis of vascular cells such as endothelial cells and smooth muscle cells is thought to be associated with pathogenesis of atherosclerosis and restenosis after balloon angioplasty. Indeed, several investigators demonstrated the presence of apoptosis in human and experimental atherosclerotic and restenotic lesions (9,10,21). Because statins have been shown to attenuate the progression of atherosclerosis and neointimal thickening after balloon angioplasty (7), we hypothesized that statins affect apoptosis of vascular endothelial cells and VSMCs. Statins (pravastatin and fluvastatin) had no effect on apoptosis of human aortic and umbilical cord vein–derived endothelial cells (data not shown). Nevertheless in VSMCs, high levels of fluvastatin itself induced apoptosis, whereas low levels of fluvastatin had no effect. Interestingly, we found that low levels of fluvastatin markedly induced apoptosis when cells were stimulated with inflammatory cytokine interleukin-1β. Guijarro et al. (22) recently reported that another lipophilic statin, atorvastatin, induced apoptosis in VSMCs and suggested a role for prenylated proteins in the regulation of VSMC apoptosis. Consistent with their study, we also showed that the mevalonate pathway and a protein prenylation play a substantial role in fluvastatin-enhanced apoptosis. Recently, Knapp et al. (23) reported that although VSMCs are normally resistant to Fas-ligand-mediated apoptosis, the cells can be sensitized to apoptosis when exposed to simvastatin or atorvastatin. Furthermore, this sensitization to apoptosis is also observed when cells are treated with inflammatory cytokines (interferon-γ alone or cytokine cocktail— interleukin-1β + tumor necrosis factor-α + interferon-γ). However, the mechanisms for the sensitization by statins have been unknown.
In contrast to fluvastatin, pravastatin does not enhance apoptosis in interleukin-1β-stimulated cells. It has been demonstrated that fluvastatin and pravastatin exert different effects on smooth muscle proliferation. We also recently reported that fluvastatin but not pravastatin upregulates inducible nitric oxide expression in VSMCs (24). A previous study revealed that pravastatin inhibited sterol synthesis in hepatocytes with potency equivalent to other statins (25). Therefore, the difference between pravastatin and fluvastatin may be related to the former's hydrophilic nature and lack of a specific carrier for pravastatin on the extrahepatic cell membrane, making diffusion of pravastatin through the plasma membrane difficult. The specific carrier of pravastatin may exist only in hepatocytes (25).
We demonstrated that the geranylgeranylation of signaling molecule(s) is involved in statin-enhanced apoptosis. Among geranylgeranylated proteins, small GTP-binding proteins are key elements in signal transduction from membrane receptors involved in proliferation and survival of VSMCs (26). In this regard, Rho may be one of the candidate molecules because Rho/Rho kinase has been recently reported to be involved in apoptosis (27). In addition, several investigations suggest that Rho is a downstream molecule in statin-induced effects in VSMCs (22,24), suggesting that Rho is a critical molecule in statin-induced apoptosis in cytokine-stimulated VSMCs.
The involvement of MAPKs including ERK1/2 and p38-MAPK in apoptosis has been demonstrated in certain cell types (19); however, the roles of MAPKs in apoptosis are controversial. For example, p38-MAPK is proapoptotic in neural cells (28), whereas it is anti-apoptotic in tumor necrosis factor-α-treated fibroblast cell lines (29), suggesting that consequences of MAPK activity vary considerably among cell types. In VSMCs, it is reported that vasoactive peptide endothelin-1 inhibits serum deprivation–induced apoptosis through the ERK1/2 pathway (30). Our findings in this study suggest that the ERK1/2 pathway plays an anti-apoptotic role in fluvastatin-enhanced apoptosis. In contrast to ERK1/2, our results suggest that the p38-MAPK pathway plays a proapoptotic role in fluvastatin-enhanced apoptosis. Although the mechanism for the effects of ERK1/2 and p38-MAPK on fluvastatin-enhanced apoptosis is complicated, our findings provide insight into the pathway of fluvastatin-enhanced apoptosis. Because fluvastatin had no effects on either ER1/2 or p38-MAPK activity, fluvastatin may act downstream of ERK1/2 and p38-MAPK. We further speculate that fluvastatin may interact with the molecules that are induced by ERK1/2 and p38-MAPK, and this interaction may be important for VSMC apoptosis. Finally, two caspase inhibitors (YVAD-FMK and DEVD-FMK) significantly inhibited fluvastatin-enhanced apoptosis, suggesting the importance of the caspase family of proteases in the apoptosis. Further investigations are required for an understanding of the precise mechanisms of fluvastatin-enhanced apoptosis in interleukin-1β-stimulated VSMCs.
In conclusion, we demonstrated that lipophilic statin (fluvastatin) but not hydrophilic statins (pravastatin) enhanced apoptosis in cytokine-stimulated VSMCs. We found that fluvastatin induces apoptosis in cytokine-stimulated VSMCs at a concentration of 1–3 μM. The peak concentration (C max ) of fluvastatin in the plasma after administration of multiple doses is 1 μM(31). Thus, clinical concentrations of fluvastatin may induce apoptosis in VSMCs and influence the vascular remodeling and function in vivo. The results from the use of isoprenoids, MAPK inhibitors, and caspase inhibitors suggest that protein prenylation, ERK1/2, p38-MAPK, and caspases play a role in the pathways of fluvastatin-enhanced apoptosis. Of note, inhibition of the ERK1/2 pathway further increases fluvastatin-enhanced apoptosis in cytokine-stimulated VSMCs, whereas inhibition of the p38-MAPK pathway prevents it. Because involvement of cytokines including interleukin-1β and tumor necrosis factor-α in atherosclerotic and restenotic lesions after balloon angioplasty has been demonstrated (32,33), the modification of the MAPK pathway with statins may become useful for the treatment of atherogenesis and restenosis after vascular injury.
This study was supported by grants from the Ministry of Education, Science, Sports and Culture, Japan (No. 40296108) and the Jichi Medical School Young Investigator Award. The authors thank Toshiko Kambe for her expert technical assistance.
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Keywords:© 2002 Lippincott Williams & Wilkins, Inc.
Apoptosis; Atherosclerosis; Cytokines; Signal transduction; Smooth muscle