The accumulation of vascular smooth muscle cells (VSMCs) within the intima is an early feature of atherosclerosis that results from an imbalance of cell proliferation and apoptosis.1,2 This imbalance may be affected by interactions with other cell types at sites of atherosclerosis, in particular inflammatory cells, which release a variety of cytokines, chemokines, and mediators of oxidative stress.3 Oxidants released by inflammatory cells within atherosclerotic lesions are thought to exert their effects via actions on VSMCs,4-6 although the precise role of oxidant stress in the progression of atherosclerosis remains unclear.
Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (statins) reportedly inhibit VSMC proliferation and induce apoptosis independently of their lipid-lowering effect.7-10 Instead, the inhibitory effects of statins on Ras and Rho3,11 and their antioxidant activity are thought to be involved.12,13 In addition, mitogen-activated protein kinase (MAPK)-dependent pathways are also thought to be involved in the intracellular signal transduction downstream of oxidative stress, given that oxidative stress leads to activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK in VSMCs.4,14,15 However, it is not well understood how statins regulate cellular processes in VSMCs exposed to oxidant stress or how they affect MAPK-dependent pathways. Also unknown is the effect of statins on the expression of MAPK phosphatase-1 (MKP-1), an important negative regulator of MAPK activity.16 More important, earlier studies were often carried out using concentrations of statins that were much higher than the therapeutic levels.8-10 Bearing all this in mind, we hypothesized that, at therapeutic levels, statins influence oxidant stress-induced apoptosis in VSMCs via modulation of MAPK signaling. To test that idea, we evaluated the effects of a therapeutic concentration of the lipophilic statin pitavastatin13,17,18 on the oxidant stress-induced apoptosis and on levels of MAPK activation, expression of MKP-1, and protein prenylation in cultured human VSMCs.
Pitavastatin was provided by Kowa Pharmaceuticals Co. Ltd. (Tokyo, Japan). H2O2 and simvastatin were purchased from Wako (Osaka, Japan). Pravastatin; xanthine (X); xanthine oxidase (XO); mevalonate; farnesylpyrophosphate (FPP); geranylgeranylpyrophosphate (GGPP); and antibodies against p38 MAPK, phosphorylated ERK (p-ERK), phosphorylated JNK (p-JNK), and phosphorylated p38 MAPK (p-p38 MAPK) were from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against ERK, JNK, and MKP-1 were from Santa Cruz (Santa Cruz, CA, USA).
Cell Culture and Treatments
Human aortic smooth muscle cells were obtained from Cascade Biologics (Portland, OR, USA) and cultured in the growth medium from Kurabo (HuMedia; Osaka, Japan) supplemented with 5% fetal bovine serum (FBS). The cells were used between passages 5 and 7, when they attained approximately 80% confluency. We initially determined the toxicity of various concentrations of H2O2 and pitavastatin on VSMCs. When orally administered to healthy volunteers at the usual dosages, the plasma concentration of pitavastatin reportedly ranges from 0.001 to 0.1 μmol/L.18 We therefore regarded 0.01 μmol/L as a therapeutic concentration of pitavastatin in the present study. We then incubated VSMCs for 24 hours in the presence of 300 μmol/L H2O2, 0.01 μmol/L pitavastatin, or both in DMEM (Dulbecco's modified Eagle's medium) supplemented with FBS. When used in combination, pitavastatin was added to the cultures 2 hours before addition of H2O2. A mixture of X (100 μmol/L) and XO (10 mU/ml), the X/XO system,5 was used instead of H2O2 in some experiments. In the other experiments, a hydrophilic statin pravastatin (0.034 μmol/L) and another lipophilic statin simvastatin (0.012 μmol/L), both approximately at the physiologic concentrations, were substituted for pitavastatin. Mevalonate (10 μmol/L), FPP (1 μmol/L), and GGPP (1 μmol/L) were used to assess the role played by protein prenylation in the responses to H2O2 and pitavastatin. Experiments were carried out at least in triplicate (n = 3 to 6 in each group).
Cell viability was determined using the MTT (3-[4,5- dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) method.19
In situ Nick-End Labeling
In situ nick-end labeling (TUNEL) assays were performed using an ApopTag kit (Chemicon; Temecula, CA, USA) principally according to the instructions of the supplier.
Annexin V Immunofluorescence
Apoptosis was also analyzed using the annexin V-FITC fluorescence microscopy kit (BD; Franklin Lakes, NJ, USA), which detects phosphatidylserine on the outer surface of the cell membrane. The incidence of immunopositive cells for TUNEL or annexin V assay was evaluated by counting cells in randomly chosen 20 high-power fields. Mammary tissue of mice was used as the positive control for both assays.
Scanning Electron Microscopy
VSMCs cultured on coverslips were fixed for 4 hours in phosphate-buffered 2.5% glutaraldehyde (pH 7.4), postfixed for 1 hour in 1% osmium tetroxide, and conventionally prepared for scanning electron microscopy (S-450; Hitachi, Tokyo, Japan).
VSMCs were exposed to the indicated reagents for the indicated intervals, then washed twice with PBS and lysed in Laemmli's buffer. The amount of protein in each lysate was determined using a BioRad protein assay kit (Hercules, CA, USA), after which 5-μg samples were heated for 5 minutes in a boiling water bath and subjected to electrophoresis on a 12.5% polyacrylamide-SDS (sodium dodecyl sulfate) slab gel. The resolved proteins were transferred to a polyvinylidene fluoride membrane in 25 mmol/L Tris-HCl, 192 mmol/L glycine, and 0.1% SDS. After blocking the membrane in 5% nonfat dry milk overnight at 4°C, it was incubated for 1 hour at room temperature with a primary antibody against ERK, JNK, p38 MAPK, p-ERK, p-JNK, p-p38 MAPK, or MKP-1 in PBS containing 0.05% (v/v) Tween 20 (T-TBS). The immunoreactive protein bands were then visualized using a peroxidase-conjugated secondary antibody, diaminobenzidine HCl, and an ECL Plus kit (Amersham Biosciences, Buckinghamshire, UK). The signals were quantified by densitometry.
Data were expressed as means ± standard error of the mean (SEM). Statistical comparisons were made using t tests or analysis of variance (ANOVA) followed by Newman-Keul's multiple comparisons test when appropriate. Values of P < 0.05 were considered significant.
Pitavastatin Enhances H2O2-Induced VSMC Apoptosis
Exposure to H2O2 dose-dependently reduced the survival rate among cultured human VSMCs (Fig. 1A). Based on these findings, we used H2O2 at a concentration of 300 μmol/L, which significantly reduced survival among VSMCs (31% reduction vs. control), for all subsequent experiments. At a concentration of 0.01 μmol/L, pitavastatin had no effect on VSMC viability (Fig. 1B); nevertheless, pretreatment with pitavastatin significantly augmented H2O2-induced VSMC death (48% reduction vs. control) (Fig. 1C). Pravastatin was found to augment the VSMC death induced by the X/XO system too (Fig. 1D). Such a cell death-promoting effect was also noted in simvastatin, but not in pravastatin, at the physiologic concentrations (Fig. 2).
H2O2 also significantly increased the incidence of TUNEL positivity among VSMCs (2.5 ± 0.3% vs. 1.0 ± 0.2% in the control, P < 0.05), which was further increased by pretreatment with pitavastatin (3.7 ± 0.5%). However, pitavastatin alone had no effect on the incidence of apoptosis among VSMCs (0.9 ± 0.2%) (Fig. 3A). Similarly, Annexin V-positivity, another index of apoptosis, was accelerated by pitavastatin in the VSMCs exposed to H2O2 (Fig. 3B). Typical of apoptosis, scanning electron microscopy revealed the presence of shrunken and/or lobulated cells and apoptotic bodies with smooth surfaces among the VSMCs exposed to H2O2, with or without pitavastatin (Fig. 3C).
Enhancement of H2O2-Induced VSMC Apoptosis by Pitavastatin Is Accompanied by Prolonged Activation of JNK and p38 MAPK
Western analysis revealed no changes in the levels of ERK, JNK, or p38 MAPK expression in VSMCs during incubation with H2O2 and/or pitavastatin for up to 24 hours (data not shown). However, levels of the phosphorylated (activated) forms of MAPKs were significantly increased within as little as 30 minutes after exposing cells to H2O2 (Fig. 4A). Levels of MAPK activation continued to be elevated after 2 hours of exposure to H2O2, but levels returned to baseline within 24 hours. By contrast, in pitavastatin-pretreated VSMCs, levels of p-JNK and p-p38 MAPK, but not p-ERK, continued to be elevated after 24 hours of treatment (Fig. 4A).
When we then examined expression levels of MKP-1, an important negative regulator of MAPK activation,16 we found them to be significantly elevated in VSMCs treated with H2O2 for 24 hours (Fig. 4B). This upregulation of MKP-1 expression was unaffected by pretreatment with pitavastatin (Fig. 4B).
Prenylation Is Necessary for Enhancement of H2O2-Induced VSMC Apoptosis by Pitavastatin
Finally, we evaluated the extent to which protein prenylation contributes to the enhancement of H2O2-induced VSMC apoptosis by pitavastatin. We found that the effect of pitavastatin was completely blocked in the presence of mevalonate, FPP, or GGPP (Fig. 5).
We have shown that pretreatment with a therapeutic concentration of pitavastatin (0.01 μmol/L) enhances H2O2-induced cell death among VSMCs, although pitavastatin alone had no effect on VSMC viability. Moreover, TUNEL assays and scanning electron microscopic analysis revealed apoptosis to be the cause of VSMC death. Pitavastatin showed the same cell death-promoting effect on VSMCs that were exposed to another oxidative stress-generating system (the X/XO system). These findings suggest that a therapeutic dose of pitavastatin reduces the numbers of VSMCs exposed to oxidant stress at sites of atherosclerosis, although actual concentration of H2O2 within atherosclerotic lesions was not determined. The fact that pitavastatin sensitizes VSMCs to H2O2-induced apoptosis is reminiscent of statins' ability to promote Fas ligand-induced cell death in VSMCs,20 although the molecular mechanisms appear to differ (see later). It is important to note, however, that statins are known to display antioxidant properties,21,22 appearing inconsistent with the present finding. However, an antioxidative effect does not necessarily mean antiapoptosis because induction of apoptosis by oxidative stress is critically regulated by their concentrations and because oxidants have various biological actions depending on the dosages (eg, even cell proliferation that is opposite to apoptosis).4,5 In addition, the dosages of statins in the previous studies are generally higher than the present one. These may make the present finding appear to be somewhat inconsistent with the previous one.
In VSMCs exposed to H2O2, the MAPKs ERK, JNK, and p38 MAPK were all activated within 30 minutes after stimulation, and this activation persisted for 2 hours before returning to baseline within 24 hours. By contrast, activation of JNK and p38 MAPK, but not ERK, was sustained for 24 hours when VSMCs were pretreated with pitavastatin. Although significant inhibition of ERK activation was previously observed in VSMCs treated with pitavastatin,23 the dosage used in that study was extraordinarily high (100 μmol/L). The JNK and p38 MAPK cascades are activated by a variety of stress-inducing stimuli and culminate with the phosphorylation and activation of transcriptional factors such as AP-1 and other cellular mediators of apoptosis.24-26 We therefore suggest that the sustained activation of JNK and p38 MAPK induced by pitavastatin seen in the present study is likely associated with the enhancement of H2O2-induced VSMC apoptosis. In addition, simvastatin, one of the other lipophilic statins, was revealed to show the same effect, whereas pravastatin, a hydrophilic statin, was not. Thus, such as apoptosis-promoting effect on VSMCs may be the common nature of lipophilic statins.
In addition, MKP-1 is a dual-specificity phosphatase that selectively inactivates tyrosine-phosphorylated MAPKs through dephosphorylation.15 In that regard, Guo et al27 proposed that tumor necrosis factor-α (TNF-α)-induced apoptosis in mesangial cells is blocked by MKP-1 through inhibition of JNK activation; moreover, H2O2 has been reported to upregulate MKP-1 in mesangial cells.28 Consistent with the latter, we observed similar upregulation of MKP-1 in VSMCs exposed to H2O2. Notably, however, this H2O2-induced increase in MKP-1 was unaffected by pretreatment with pitavastatin. Collectively, then, our findings suggest that the enhancement of H2O2-induced VSMC apoptosis pitavastatin is likely the result of sustained activation of JNK and p38 MAPK and is independent of MKP-1 expression. One, however, has reported involvement of Jak2 tyrosine kinase in oxidative stress-induced apoptosis in VSMCs.29 Further studies are warranted to investigate the relationship between the MAPK cascade and Jak/Stat pathway and relative importance between them in oxidative stress-induced apoptosis.
Protein prenylation is often required for proteins (eg, small GTP-binding proteins) to anchor to the cell membrane and fully function as signal transducers.30,31 Mevalonate and its derivatives, FPP and GGPP, isoprenylate membrane proteins (eg, Ras) to activate their downstream signaling molecules (eg, Rho and MAPKs).32 Production of these prenylation inhibitors is attenuated by statins, and high concentrations of several lipophilic statins have been shown to inhibit Rho prenylation, thereby inducing apoptosis in cultured VSMCs.8 We therefore evaluated the extent to which protein prenylation contributes to the enhancement of H2O2-induced VSMC apoptosis by pitavastatin. That treating cells with mevalonate, FPP or GGPP completely blocked the effect of pitavastatin suggests the enhancement of H2O2-induced VSMC apoptosis by pitavastatin is dependent on protein prenylation, although the prenylated proteins remain to be determined.
At a therapeutic dosage pitavastatin promotes oxidant stress-induced VSMC apoptosis, and it is suggested that the molecular mechanism involves activation of MAPKs and protein prenylation. This finding may explain, at least in part, the regressive effect of statins on atherosclerotic lesions, which are often surrounded by an oxidative milieu.
We thank Hatsue Oshika and Kazuko Goto for technical assistance.
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