Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10236
RESEARCH ARTICLE
Simvastatin promotes cardiac microvascular endothelial cells proliferation, migration and survival by phosphorylation of p70 S6K and FoxO3a Qiao Pan1y, Xiaobo Xie2y, Yan Guo3 and Haichang Wang1* 1 Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710032, China 2 Department of Disease Surveillance and Control, Centers for Diseases Control and Prevention of Guangzhou Military District, Guangzhou 510507, China 3 Department of Oncology, State Key Discipline of Cell Biology, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710032, China
Abstract Restenosis severely limits the overall efficacy of interventions. One of the reasons is the lack of reendothelialization related to inhibition of endothelial cell proliferation and migration since drug is delivered to the luminal surface. Statins can promote angiogenic processes by improving endothelial function, proliferation and migration in cardiac microvascular endothelial cells (CMECs). This study clarified the effect of simvastatin on Akt/mTOR/p70 S6K and FoxO3a signalling pathways in rat CMECs following pretreated with rapamycin. Rapamycin treatment for 24 h inhibited CMECs’ proliferation, migration and NO (nitric oxide) secretion, but with increased cell apoptosis and reactive oxygen species (ROS) production. In contrast, simvastatin pretreatment significantly improved proliferation, migration and NO secretion, and inhibited CMECs’ apoptosis and ROS production in rapamycin-induced CMECs. Western blot assay showed that, after treatment with simvastatin, the phosphorylation of Akt/mTOR/p70 S6K and FoxO3a were up-regulated in rapamycin-induced CMECs, which was significantly reversed by pretreatment with LY294002. The data suggest that simvastatin inhibits rapamycin-induced CMECs dysfunction and apoptosis, probably through activation of PI3K/Akt/mTOR/p70 S6K and mTOR/FoxO3a signalling pathway in a sequential manner and this pathway may be important in some of the pleiotropic effects of statins. Keywords: simvastatin; cardiac microvascular endothelial cell; Akt; FoxO3a; nitric oxide (NO); oxidative stress
Introduction Restenosis severely limits the overall efficacy of interventions and can occur in up to 80% of patients (Clowes and Reidy, 1991). Although Cypher rapamycin-eluting stents and Taxus paclitaxel-eluting stents significantly reduce the risk of target vessel revascularisation compared to bare metal stents (BMS). Late stent thrombosis after Cypher and Taxus drugeluting stent (DES) placement still has emerged as a major concern (Finn et al., 2007). The process involves a complex cascade of reactions that result in luminal narrowing through a combination of neointimal hyperplasia and constrictive
remodelling. The limitation of DES is the lack of reendothelialization related to inhibition of endothelial cell proliferation and migration since drug is delivered to the luminal surface (Finn et al., 2007; Seedial et al., 2013). 3Hydroxy-3-methyl-glutaryl-CoA reductase inhibitors, statins, are potent cholesterol-lowering drugs. Recently, in vivo and in vitro studies have demonstrated that, the drugs have additional vasculo-protective effects independent of cholesterol lowering: statins improve endothelial function, reduce vascular inflammation, decrease platelet aggregation and enhance endothelial processes involved in angiogenesis (Undas et al., 2005; Fadini et al., 2010). Moreover, statins
Corresponding author: e-mail: [email protected] These authors contributed equally to this work. Abbreviations: CMECs, cardiac microvascular endothelial cells; DES, drug-eluting stent; DHE, staining dihydroethidine; eNOS, endothelial nitric oxide synthase; FoxO, the ForkheadBox class O; mTOR, mammalian target of rapamycin; MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide]assay; NO, nitric oxide; p70S6K, p70 S6 kinase; PI3Ks, phosphoinositide 3-kinases; ROS, reactive oxygen species; WST-8, [2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium, monosodium salt] viability assay
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promote angiogenic processes, including CMECs proliferation and migration (Weis et al., 2002; Katsumoto et al., 2005) and exert direct beneficial effects on the endothelium in part through an increase in nitric oxide (NO) production (Brouet et al., 2001). However, the molecular mechanism underlying these serum lipid-independent effects of statins in CMECs is not well understood yet. It is well established that activation of phosphatidylinositol 3-kinase (PI3K) plays an important role in promoting cell survival and proliferation in numerous cell systems (Ruckerl et al., 2012; Thomas et al., 2013). And also as an essential role in angiogenesis (Yoshioka et al., 2012). In CMECs, it has been shown that the majority of growth factor-induced responses are mediated by the activation of the PI3K/Akt signalling cascade (Somanath et al., 2006) and statins could rapidly induce the activation of Akt in these cells (Brouet et al., 2001). Simvastatin has been shown to reduces venous stenosis formation in amurine hemodialysis vascular access model (Janardhanan et al., 2013). We have directed our attention to the molecular mechanisms by which simvastatin induces proliferation and migration in CMECs. We found that CMECs proliferation, migration and survival were enhanced by simvastatin, and this effect was mediated by PI3K/Akt-induced mammalian target of rapamycin (mTOR)/p70 S6 kinase (p70 S6K) and mTOR/FoxO3a activation in rat CMECs. Materials and methods
Animals Twenty male SD (Sprague–Dawley) rats (100–150 g) were purchased from the Experimental Animal Center of the Fourth Military Medical University. All animal procedures were performed in accordance with protocols approved by the Fourth Military Medical University Animal Research Committee and International Research Animal Care Guidelines.
Isolation, cultivation and identification of CMECs Briefly, CMECs were enzymatically released from left ventricular tissue of adult male SD rats using 0.2% collagenase type II (Worthington Biochemical Corp., Lakewood, NJ, USA) in sterile Hank’s buffer solution (HBSS) (Invitrogen, LuBio Science, Lucerne, Switzerland) for 20 min at 378C followed by 0.25% trypsin (Invitrogen) for 20 min at 378C in a shaking water bath according to the method developed by Nishida et al. (1993) with minor modifications. CMECs reached 80% confluence within 3–4 days in DMEM (Amimed, BioConcept, Allschwil, Switzerland) with 20% new-born calf serum (NCS) (Invitrogen, USA) and antibiotics. No additional growth factors were added to the growth medium. CMEC were then used in 600
the second passage and starved for 1 day in DMEM with 1% NCS before pharmacologic treatments. Cultured CMECs were identified by morphological and functional characteristics (Wei et al., 2010) with microscopy and Dil-ac-LDL intake assay (Molecular Probes, Eugene, OR, USA). Experiments were performed on cells at passage three to five from primary culture.
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide] assay CMECs were plated overnight in 96-well tissue culture plastic dishes at 1 104 cells/mL. Cells were treated with (1) rapamycin (Calbiochem, San Diego, CA, USA) at 0, 102, 101, 100, 101 nM or (2) rapamycin (100 nM) in the absence or the presence of simvastatin (Sigma–Aldrich, USA) at 0, 102, 101, 100, 101 mM for an additional 24 h. Cells from each group were harvested and 100 mL MTT (Sigma– Aldrich) was added into each well and incubated for 4 h. At the end of this treatment, the incubation medium was removed and formazan crystals were dissolved in 100 mL DMSO. MTTreduction was quantified by measuring the light absorbance at 490 nm.
WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] viability assay CMECs were plated into 96-well plates until 80% confluence then starved with DMEM 0.5% NCS for 24 h and treated in line with the MTT assay. CMECs were incubated for 2 h at 378C with WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] (CCK-8) (Dojindo, Labforce, Nunningen, Switzerland) while they released orange formazan into the medium and absorption was measured using a Safire multiwell-plate reader (Tecan). The WST-8 assay does not include washing steps and therefore is more suitable for stressed CMECs that tend to detach and get lost during the assay.
Cell migration assay For transwell migration assays, the cells were placed into the upper chamber on uncoated membranes (24-well insert; pore size 8 mm; BD Biosciences, California, USA). The cells were incubated for 24 h, and cells that did not migrate through the pores were removed using a cotton swab. The cells on the lower surface of the membrane were stained with gentian violet for 10 min and counted. Migration was assessed by counting cells in five microscopic fields per well at 400 magnification. The data are representative of triplicate experiments. Cell Biol Int 38 (2014) 599–609 ß 2013 International Federation for Cell Biology
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Assessment of apoptosis of CMECs
NO measurement
Following pretreatment for 24 h, CMECs were fixed, stained with Hochest 33258 (Sigma–Aldrich) and viewed by fluorescence microscopy (Olympus, Tokyo, Japan). Cells were designated as apoptosis with highly condensed, brightly staining nuclei and non-apoptosis with light blue staining. The apoptotic index was defined as the ratio of the apoptotic cell number to total cell number. Caspase-3 activity was determined by the Caspase-Activity Assay Kit (Chemicon, Billerica, USA).
The amount of NO production released by CMECs was determined by measuring the concentration of nitrite, a metabolite of NO, with a modified Griess reaction method (Nanjing Jiancheng Institute of Biological Engineering). Briefly, CMECs were seeded at 6 105 cells/well on a six-well plate one day before the treatment. Adherent cells were pretreated with rapamycin and/or LY294002 for 1 h and then with simvastatin for a further 24 h. Finally, supernatant was collected and mixed with an equal volume of modified Griess reagent for the colorimetric assay. After 10 min incubation at room temperature, the concentration of the resultant chromophore was measured spectrophotometrically at 550 nm after enzymatic conversion of the supernatant nitrate to nitrite by nitrate reductase. The nitrite concentration in the samples was calculated from nitrite standard curves made from sodium nitrite using the same culture medium.
Western blot analysis To determine the role of PI3K pathway, in addition to previous groups, we have added a group of rapamycin (100 nM) in the absence or the presence of simvastatin (100 mM) and LY294002 (Calbiochem; the inhibitor of PI3K) at 50 mM. Cells from each group were cultured and harvested at the indicated time. Cells were washed three times with cold PBS, and scraped off using an ice-cold lysis buffer. Proteins were prepared and separated on 10% SDS–PAGE gels before being transferred electrophoretically to nitrocellulose membranes. After blocking with 5% (w/v) non-fat dried skimmed milk powder for 1 h, the membranes were immunoblotted with the appropriate primary antibody at 48C overnight. The membranes were washed and further incubated with the secondary antibody at 378C for 60 min. Blots were visualized using an ECL (enhanced chemiluminescence system, Amersham). The following primary antibodies were used: Akt, phosphor-Akt (Ser73), FoxO3a, phospho-FoxO3a (Thr32), p70S 6K, phospho-p70S 6K (Thr389). Anti-bodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
Real-time reverse transcriptase PCR Real-time PCR assays were used on the samples to measure the expression of eNOS (QT0010075) and NOS2 (NM 010927). For gene analysed, reactions involved 1 mL Qiagen Quanti Tect Primer Assay added to 5 mL Power SyBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) and diluted with 2 mL molecular grade water. A final volume of 8 mL was dispensed into each well and 2 mL diluted cDNA (160 ng/reaction) was added. Each sample was tested in triplicate for each gene, and PCR reactions were performed using ABI Prism 7900 real-time PCR equipment. The thermal profile consists of 958C for 15 min, then 40 cycles of 948C for 15 s, 558C for 30 s and 728C for 30 s. This was plotted as a melting curve. The comparison between samples was performed using GAPDH and RPL32 as internal standards. REST MCS software was utilized for the calculation of the relative differences between the test groups. Cell Biol Int 38 (2014) 599–609 ß 2013 International Federation for Cell Biology
Quantification of ROS production in CMECs ROS production, an index of oxidative stress, in viable CMECs was measured by lucigenin-enhanced chemiluminescence assay adapted from the method (Schuhmacher et al., 2010). ROS production was expressed as relative light units (RLU) per second per million cells (RLU/s/million cells). Dihydroethidine (DHE; Beyotime, China) staining was used to detect the in situ formation of superoxide according to the oxidative fluorescent microtopography, as described recently (Zuo et al., 2011). DHE staining was visualized under a confocal microscope (Olympus). Images of cells were analysed with Image-Pro Plus software version 6.0. The mean fluorescence intensity of each cell was calculated, and the total cell emission signals per field were averaged for data analysis.
Statistical analysis All values were presented as means SD of n independent experiments. Most data (except Western blot density) were compared by ANOVA followed by Bonferroni correction for post hoc analysis. P < 0.05 was considered statistically significant. GraphPad Prism software version 5.0 (GraphPad Software) was used for the calculations. Results
Identification of CMECs The cells had ‘flagstone’ morphology (Figure 1A-a), and tested positive with the Dil-ac-LDL intake assay (Figure 1Ab), both indicating that the cultured cells were CMECs. 601
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Figure 1 Characterization of CMECs and to detected the proliferation and migration in rapamycin-induced CMECs. (A-a) CMECs monolayer presents cobble stone appearance by phase-contrast microscopy (scale bar: 40 mm); (A-b) Uptake of Dil-Ac-LDL by immunofluorescence (red, Dil-Ac-LDL; blue, DAPI, scale bar: 50 mm); CMECs were exposed to rapamycin for 24 h and proliferation was measured by the MTT-assay (B) and WST-8 assay (C). Representative image (D) of membranes where the lower well cell medium contained different concentration of rapamycin (scale bar: 550 mm); (E) Quantity of migration number of CMECs detected by transwell assays. P < 0.05, #P < 0.01 and &P < 0.001 versus control. Each column represents the mean SD of six different experiments. Abbreviations: Con, control; NS, no significant.
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Rapamycin suppresses CMECs proliferation and migration in a dose-dependent manner CMECs proliferation is a crucial process for angiogenesis. The MTT assay was used to investigate whether rapamycin is capable of restricting CMECs proliferation. Treated with the increasing concentrations of rapamycin for 24 h, the viability of CMECs was decreased in a dose-dependent manner (Figure 1B). Compared to the MTT assay, the WST-8 assay showed similar results in CMECs, with a significant reduction at 100 and 101 nM of rapamycin, respectively (Figure 1C). Migration assays were done with the transwell system. Consistent with its effects on proliferation, rapamycin also clearly suppressed CMECs migration (Figures 1D and 1E). Based on the results, we chose a concentration 100 nM for further study.
Protection of simvastatin in CMECs
the expression of caspase-3 activity and found that, simvastatin statistically decreased the caspase-3 expression in rapamycin-induced CMECs (Figure 2G). These results show that simvastatin had an anti-apoptotic effect on rapamycin-induced CMECs.
Simvastatin induces p70 S6K, FoxO3a phosphorylation via PI3K/Akt pathways in CMECs
To investigate whether simvastatin is capable of promoting CMEC proliferation, CMECs were then treated with vehicle or simvastatin (100 mM) only as control. After pretreated with rapamycin (100 nM), the cell viability in CMECs treated with different concentrations of simvastatin was significantly higher than that in rapamycin group by MTT assay (Figure 2A), especially treated with simvastatin at the concentrations 101 or 100, 101 mM. However, there was no significant difference between 100 and 101 mM simvastatin group. The WST-8 assay results (Figure 2B) were similar with MTTassay. The cell migration assays showed that supplement of simvastatin could improve the number of migrated cells into the lower chamber after pretreated with rapamycin (Figures 2C and 2D). Both of these data indicated that simvastatin at 100 mM significantly enhanced the viability and migration of CMECs after pretreated with rapamycin. However, there was no further effect of simvastatin administration at higher concentrations of 101 mM on the viability and migration of CMECs after pretreated with rapamycin.
In CMECs, the majority of growth factor-induced responses are mediated by the activation of PI3K/Akt signalling pathway (Somanath et al., 2006). These results prompted us to further investigate the mechanisms involved in the improvement of simvastatin after rapamycin-induced injury. We analysed whether simvastatin activates this pathway in CMECs. Cells were stimulated with 100 mM simvastatin for 24 h and cell lysates were prepared and subjected to Western blot analysis. Addition of simvastatin resulted in phosphorylation of Akt (Figures 3A and 3B). We tested the effect of simvastatin on the phosphorylation of a PI3K/Akt downstream target, p70 S6K. p70 S6K helps regulate the translation machinery and is controlled by mTOR, an immediate downstream effector of Akt (Miriuka et al., 2006). Simvastatin induced the phosphorylation of p70 S6K (Figures 3A and 3C), thereby leading to enzyme activation. Pretreatment of cells with LY294002, a specific PI3K inhibitor, abolished the phosphorylation of Akt and p70 S6K, whereas pretreatment with rapamycin, a specific inhibitor of mTOR, inhibited only p70 S6K activation (Figures 3A–3C). Thus simvastatin activates PI3K/Akt/mTOR/p70 S6K signalling in a sequential manner in CMECs. The Forkhead Box, class O (FoxO) family of Forkhead transcription factors, is a regulator of oxidative stress (Salih and Brunet, 2008). These transcription factor are also downstream targets of mTOR (Tesio et al., 2011) and are inactivated by Akt-dependent phosphorylation of serine/ threonine residues (Birkenkamp and Coffer, 2003). We investigated whether simvastatin phosphorylates and thereby inactivates the forkhead transcription factors. Simvastatin also phosphorylated FoxO3a with the same kinetics as for Akt and p70 S6K (Figures 3A and 3D).
Simvastatin prevented rapamycin-induced apoptosis of CMECs
Simvastatin promotes the secretion of NO in CMECs via a PI3K/Akt dependent mechanism
To investigate the role of simvastatin on rapamycin-induced apoptosis in CMECs, Hochest 33258 staining assay was performed. Rapamycin induced a significant increase in Hochest 33258 staining positive cells as brightly stained nuclei. Compared with cells cultured in rapamycin, each simvastatin treated group showed a significant decrease in apoptotic cells (Figures 2E and 2F), especially in the concentrations of simvastatin at 100 mM. We also detected
Rapamycin (100 nM) significantly decreased NO secretion in comparison with the control group (21.7 2.9 mmol/L vs. 36.3 5.8 mmol/L, P < 0.01; Figure 4A). However, pretreatment with simvastatin (100 mM) enhanced the NO secretion both in basal (46.7 7.3 mmol/L vs. 36.3 5.8 mmol/L, P < 0.01) and in rapamycin-induced groups (35.1 3.6 mmol/L vs. 21.7 2.9 mmol/L, P < 0.01). Real time PCR analysis showed that rapamycin downregulated the
Simvastatin significantly improves the proliferation and migration in CMECs
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Figure 2 Effect of simvastatin on CMECs’ proliferation, migration and apoptosis. CMECs were treated with rapamycin in the absence or the presence of simvastatin. Viable cell numbers were monitored by MTT assay (A) and WST assay (B); Representative image (C, scale bar: 550 mm) and quantity (D) of migration detected by transwell assay. CMECs were stained with Hochest 33258 and observed by fluorescence microscopy. Representative image (E, scale bar: 50 mm) and quantity (F) of apoptosis by simvastatin treated. (G) Expression of caspase-3 activity assay was quantified. Each column represents the mean SD of six different experiments. P < 0.05, versus control, #P < 0.05 versus rapamycin (100 nM) alone group, &P < 0.05 versus simvastatin at 100 mM group. Abbreviations: Con, control; S, simvastatin; R, rapamycin; NS, no significant.
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Figure 3 Effect of simvastatin induces phosphorylation of Akt, p70 S6K and FoxO3a in a PI3K-dependent manner. Western blotting was performed to analyse the phosphorylation of Akt, p70 S6K and FoxO3a. (A) Representative blot. Quantitative densitometry data of pAkt (B), p-p70 S6K (C) and pFoxO3a (D) expression of CMECs in different groups. As a loading control, the membranes were stripped and reprobed with antibodies for the total level of each protein. Each column represents the mean SD of six different experiments. P < 0.01, versus control, #P < 0.05 versus rapamycin (100 nM) alone group, &P < 0.01 versus rapamycin (100 nM) and simvastatin (100 mM) and LY294002 (50 mM) group. Abbreviations: Con, control; S, simvastatin; R, rapamycin; LY, LY294002, Akt inhibitor.
expression of eNOS mRNAs in CMECs (P < 0.05), whereas pretreatment with simvastatin upregulated the expression (Figure 4B, P < 0.05). The data indicated that simvastatin pretreatment increased NO release in both basal and rapamycin-induced groups by increasing the expression of eNOS. Both of these effects, including NO secretion and eNOS expression, were attenuated with pretreatment with LY294002.
cence assay (Figure 5C). Both suggested that exposure of CMECs to rapamycin promoted intracellular ROS levels, which was attenuated by simvastatin. However, the inhibitory effect of simvastatin on rapamycin-induced ROS production was abolished by LY294002. Thus simvastatin might decrease rapamycin-induced ROS accumulation in CMECs through PI3K/Akt signalling pathway. Discussion
Simvastatin decreases rapamycin-induced ROS production in CMECs To determine the mechanism of action behind the beneficial effect of simvastatin on CMECs, ROS production was measured by two independent approaches: DHE staining (Figures 5A and 5B) and lucigenin-enhanced chemiluminesCell Biol Int 38 (2014) 599–609 ß 2013 International Federation for Cell Biology
Without exception, all interventions designed to treat atherosclerotic occlusive disease are complicated by restenosis (Seedial et al., 2013). Despite many advances in the fields of vascular biology, pharmacology and bioengineering, restenosis remains a significant problem (Clowes et al., 1983a, b). One of the reasons causing restenosis is 605
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Figure 4 Effects of simvastatin on NO release and eNOS expression in rapamycin-induced CMECs. Simvastatin significantly increases NO secretion (A) both in basal and in rapamycin-induced groups. (B) eNOS mRNA data. This improvement (including NO secretion and eNOS expression) by simvastatin could attenuate with pretreatment with LY294002. Each column represents the mean SD of six different experiments. P < 0.05, versus control, #P < 0.05 versus rapamycin alone group, &P < 0.01 versus rapamycin (100 nM) and simvastatin (100 mM) and LY294002 (50 mM) group. Abbreviations: Con, control; S, simvastatin; R, rapamycin; LY, LY294002, Akt inhibitor.
related to inhibition of endothelial cell proliferation and migration since drug is delivered to the luminal surface from DES (Finn et al., 2007; Seedial et al., 2013). All forms of angiogenesis may share certain basic features, including
CMECs proliferation and migration, etc. (Carmeliet, 2003) and are regulated by many pro-angiogenic growth factors (Suri et al., 1996). Other than these growth factors (such as VEGF etc.), simvastatin (Shen et al., 2011) and rosuvastatin
Figure 5 Effects of simvastatin on rapamycin-induced oxidative stress in CMECs. (A) Representative diagram showing DHE staining of CMECs (red, DHE; blue, DAPI). (B) The average fluorescence intensity from five fields was summarized. (C) Superoxide generation of CMECs in different groups. Each column represents the mean SD of six different experiments. Scale bar: 40 mm. P < 0.01, versus control, #P < 0.05 versus rapamycin (100 nM) alone group, &P < 0.05 versus rapamycin (100 nM) and simvastatin (100 mM) and LY294002 (50 mM) group. Abbreviations: Con, control; S, simvastatin; R, rapamycin; LY, LY294002, Akt inhibitor; DHE, dihydroethidine.
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(Zhou et al., 2013) have an angiogenic effect on CMECs. CMECs proliferation is a crucial process for angiogenesis. Cerivastatin and pitavastatin stimulate CMECs proliferation (Weis et al., 2002; Katsumoto et al., 2005). Statins have an anti-apoptotic effect on endothelial progenitor cells (Ruckerl et al., 2012). Therefore, in investigating whether simvastatin promotes CMECs proliferation, migration and survival after pretreated with rapamycin, we found that low concentration (100 mM) of simvastatin could significantly improve proliferation, migration and apoptosis in CMECs, whereas higher concentrations (101 mM) offered no further improvement consistent Katsumoto et al. (2005). The results confirmed that, at therapeutic doses, statins promoted proliferation, migration and survival of CMECs. Phosphorylation of Akt targets may contribute to CMECs functioning, including survival, growth and migration (Somanath et al., 2006). Activation of Akt promotes endothelial cell survival by inhibiting apoptosis (Gerber et al., 1998; Dimmeler et al., 2001), stimulating endothelial NO synthesis (Dimmeler et al., 1999). mTOR is an important downstream target of PI3K/Akt, and mTOR signalling is involved in the control of cell growth and proliferation (Sarbassov et al., 2005a). mTORC1 activation leads to phosphorylation of p70 S6K, and mTORC1 signalling is inhibited by rapamycin (Jacinto et al., 2004). mTORC1 is the downstream target of the Akt and the PI3K/Akt/mTOR/p70 S6K pathway assists in the control of cell growth and proliferation (Hay and Sonenberg, 2004; Sarbassov et al., 2005a). The role of the mTOR pathway involved in rapamycin-induced CMECs injury is not fully understood. Many studies (Jacinto et al., 2004; Sarbassov et al., 2005a; Zhang et al., 2005; Li et al., 2007) indicate that the mTOR signalling pathway is important in angiogenesis. To demonstrate that Akt is indeed an essential down-stream signalling event, Akt phosphorylation (indicative of Akt activity) was detected by Western blotting. Simvastatin also robustly activates the Akt/p70 S6K pathway in CMECs, and this activation is completely inhibited by LY294002. Whereas pretreatment with rapamycin, a specific inhibitor of mTOR, inhibits only p70 S6K activation. Because PI3K has many downstream targets that are essential for cell survival, it is possible that inhibition of PI3K leads to cell death and thereby accounts for the decrease in the CMECs’ proliferation, migration and survival. On the other hand, rapamycin inhibits both basal and simvastatin-induced p70 S6K activity, but the amount of suppression is smaller than that treated by PI3K inhibition. These results raise the possibility that simvastatin enhances angiogenesis via the PI3K/Akt/mTOR/ p70 S6K signalling pathway (Figure 6). Iantorno et al. (2007) found that PI3K/Akt signalling pathway is involved in production of NO in CMECs. NO is the most important vasodilator (Moncada and Higgs, 2006), and rapamycin can inhibit NO secretion in a dose-dependent Cell Biol Int 38 (2014) 599–609 ß 2013 International Federation for Cell Biology
Protection of simvastatin in CMECs
Figure 6 Proposed scheme for protection effect by simvastatin in CMECs. Simvastatin activates PI3K/Akt/mTOR pathways, which inactivate FoxO3a and p70 S6K by phosphorylation. FoxO3a, members of the FoxO (Forkhead box, class O) subfamily of forkhead transcription factors is a regulator of oxidative stress and p70S6K, a key translation initiation and elongation regulator, both of them can be phosphorylated by mTOR. Subsequently, FoxO3a inhibition by rapamycin and LY294002 causes increased ROS production. Rapamycin can inhibit NO secretion directly. Simvastatin increased levels of PI3K/Akt/mTOR phosphorylation, which improve the CMEC proliferative, migration and anti-apoptosis after rapamycin-induction.
manner (Miriuka et al., 2006). We found that simvastatin pretreatment increased eNOS activity, thus mediating the upregulation of NO in CMECs. We also showed that the effect of simvastatin is closely related to the PI3K/Akt activity. LY294002, a highly selective PI3K inhibitor, almost completely inhibited simvastatin-induced Akt phosphorylation in CMECs and markedly suppressed simvastatininduced CMECs secretion of NO. Pretreatment with rapamycin inhibited only the activation of mTOR downstream molecules and had little effect on NO secretion. These results suggest that the effect of PI3K activation of NO secretion in CMECs is mediated in part by the mTOR pathway, which could be the mechanism behind its favourable effects on neovascularization. The apoptosis of CMECs may be in part due to other molecules that are not related to PI3K/Akt/mTOR/p70 S6K signalling. FoxOs are a direct substrate of the protein kinase 607
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Akt, a mammalian target of rapamycin inhibition (mTOR) target (Sarbassov et al., 2005b), which inactivates them by phosphorylation (Brunet et al., 1999). Activation of Akt signalling cascades mediated anti-apoptotic and pro-proliferative signals by causing FoxO3a phosphorylation, inactivation and consequent cytoplasmic entry (Yang et al., 2008) and FoxO3a negatively regulates an anti-apoptotic microRNA (Wang and Li, 2010). Since phosphorylation determines the nuclear/cytoplasmic location of FoxO3a activity, we also tested whether simvastatin could induce the phosphorylation of FoxO3a; it activated the mTOR/FoxO3a pathway in CMECs, which was inhibited by both LY294002 and rapamycin. c-Met activation induced mTOR signalling, downregulates FoxO3a levels and ultimately increases ROS production (Tesio et al., 2011). Low levels of ROS are indispensable in many biochemical processes, including intracellular signalling, defence against microorganisms, and cell functioning (Go and Jones, 2011). In contrast, high dose and/or inadequate removal of ROS results in oxidative stress, which has been implicated in severe pathophysiological consequences (Fattman et al., 2003). Through the detection of ROS production, we found that simvastatin decreased rapamycin-induced ROS production, but did not markedly affect basal ROS production. This improvement can be abrogated by PI3K inhibition. Our data suggest mTOR/ FoxO3a/ROS signalling cascade, which decreases ROS levels, may be a part of beneficial effects of simvastatin. After pretreatment with LY294002, the significant increase of ROS production may be in part due to other molecules that are not only related to PI3K/Akt/mTOR/FoxO3a signalling (Figure 6). In summary, simvastatin has a biphasic effect on CMEC proliferation, migration and anti-apoptosis. Low concentrations of simvastatin can induce proliferation, migration and anti-apoptosis via PI3K/Akt/mTOR/p70 S6K and mTOR/FoxO3 signalling pathways, and then exert a beneficial effect by adjustment of NO and ROS production in CMECs. The possibility is raised that the Akt/mTOR pathway is important in some of the pleiotropic effects of statins.
Acknowledgement and funding The National Natural Science Foundation of China (no. E011002) supported this work.
Author contribution Qiao Pan, Xiaobo Xie, Haichang Wang, Yan Guo conceived and designed the experiments; Qiao Pan, Xiaobo Xie, Haichang Wang, Yan Guo performed the experiments; Qiao Pan, Xiaobo Xie,Yan Guo analysed the data and
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Somanath PR, Razorenova OV, Chen J, Byzova TV (2006) Akt1 in endothelial cell and angiogenesis. Cell Cycle 5(5): 512–8. Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN, Yancopoulos GD (1996) Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87(7): 1171–80. Tesio M, Golan K, Corso S, Giordano S, Schajnovitz A, Vagima Y, Shivtiel S, Kalinkovich A, Caione L, Gammaitoni L, Laurenti E, Buss EC, Shezen E, Itkin T, Kollet O, Petit I, Trumpp A, Christensen J, Aglietta M, Piacibello W, Lapidot T (2011) Enhanced c-Met activity promotes G-CSF-induced mobilization of hematopoietic progenitor cells via ROS signaling. Blood 117(2): 419–28. Thomas D, Powell JA, Green BD, Barry EF, Ma Y, Woodcock J, Fitter S, Zannettino AC, Pitson SM, Hughes TP, Lopez AF, Shepherd PR, Wei AH, Ekert PG, Guthridge MA (2013) Protein kinase activity of phosphoinositide 3-kinase regulates cytokinedependent cell survival. PLoS Biol 11(3): e1001515. Undas A, Brummel-Ziedins KE, Mann KG (2005) Statins and blood coagulation. Arterioscler Thromb Vasc Biol 25(2): 287– 94. Wang K, Li PF (2010) Foxo3a regulates apoptosis by negatively targeting miR-21. J Biol Chem 285(22): 16958–66. Wei L, Yin Z, Yuan Y, Hwang A, Lee A, Sun D, Li F, Di C, Zhang R, Cao F, Wang H (2010) A PKC-beta inhibitor treatment reverses cardiac microvascular barrier dysfunction in diabetic rats. Microvasc Res 80(1): 158–65. Weis M, Heeschen C, Glassford AJ, Cooke JP (2002) Statins have biphasic effects on angiogenesis. Circulation 105(6): 739–45. Yang JY, Zong CS, Xia W, Yamaguchi H, Ding Q, Xie X, Lang JY, Lai CC, Chang CJ, Huang WC, Huang H, Kuo HP, Lee DF, Li LY, Lien HC, Cheng X, Chang KJ, Hsiao CD, Tsai FJ, Tsai CH, Sahin AA, Muller WJ, Mills GB, Yu D, Hortobagyi GN, Hung MC (2008) ERK promotes tumorigenesis by inhibiting FOXO3a via MDM2-mediated degradation. Nat Cell Biol 10(2): 138–48. Yoshioka K, Yoshida K, Cui H, Wakayama T, Takuwa N, Okamoto Y, Du W, Qi X, Asanuma K, Sugihara K, Aki S, Miyazawa H, Biswas K, Nagakura C, Ueno M, Iseki S, Schwartz RJ, Okamoto H, Sasaki T, Matsui O, Asano M, Adams RH, Takakura N, Takuwa Y (2012) Endothelial PI3K-C2alpha, a class II PI3K, has an essential role in angiogenesis and vascular barrier function. Nat Med 18(10): 1560–9. Zhang B, Cao H, Rao GN (2005) 15(S)-hydroxyeicosatetraenoic acid induces angiogenesis via activation of PI3K-Akt-mTORS6K1 signaling. Cancer Res 65(16): 7283–91. Zhou J, Cheng M, Liao YH, Hu Y, Wu M, Wang Q, Qin B, Wang H, Zhu Y, Gao XM, Goukassian D, Zhao TC, Tang YL, Kishore R, Qin G (2013) Rosuvastatin Enhances Angiogenesis via eNOSDependent Mobilization of Endothelial Progenitor Cells. PLoS ONE 8(5): e63126. Zuo L, Youtz DJ, Wold LE (2011) Particulate matter exposure exacerbates high glucose-induced cardiomyocyte dysfunction through ROS generation. PLoS ONE 6(8): e23116. Received 2 July 2013; accepted 10 December 2013. Final version published online 10 January 2014.
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RESEARCH ARTICLE
Simvastatin promotes cardiac microvascular endothelial cells proliferation, migration and survival by phosphorylation of p70 S6K and FoxO3a Qiao Pan1y, Xiaobo Xie2y, Yan Guo3 and Haichang Wang1* 1 Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710032, China 2 Department of Disease Surveillance and Control, Centers for Diseases Control and Prevention of Guangzhou Military District, Guangzhou 510507, China 3 Department of Oncology, State Key Discipline of Cell Biology, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710032, China
Abstract Restenosis severely limits the overall efficacy of interventions. One of the reasons is the lack of reendothelialization related to inhibition of endothelial cell proliferation and migration since drug is delivered to the luminal surface. Statins can promote angiogenic processes by improving endothelial function, proliferation and migration in cardiac microvascular endothelial cells (CMECs). This study clarified the effect of simvastatin on Akt/mTOR/p70 S6K and FoxO3a signalling pathways in rat CMECs following pretreated with rapamycin. Rapamycin treatment for 24 h inhibited CMECs’ proliferation, migration and NO (nitric oxide) secretion, but with increased cell apoptosis and reactive oxygen species (ROS) production. In contrast, simvastatin pretreatment significantly improved proliferation, migration and NO secretion, and inhibited CMECs’ apoptosis and ROS production in rapamycin-induced CMECs. Western blot assay showed that, after treatment with simvastatin, the phosphorylation of Akt/mTOR/p70 S6K and FoxO3a were up-regulated in rapamycin-induced CMECs, which was significantly reversed by pretreatment with LY294002. The data suggest that simvastatin inhibits rapamycin-induced CMECs dysfunction and apoptosis, probably through activation of PI3K/Akt/mTOR/p70 S6K and mTOR/FoxO3a signalling pathway in a sequential manner and this pathway may be important in some of the pleiotropic effects of statins. Keywords: simvastatin; cardiac microvascular endothelial cell; Akt; FoxO3a; nitric oxide (NO); oxidative stress
Introduction Restenosis severely limits the overall efficacy of interventions and can occur in up to 80% of patients (Clowes and Reidy, 1991). Although Cypher rapamycin-eluting stents and Taxus paclitaxel-eluting stents significantly reduce the risk of target vessel revascularisation compared to bare metal stents (BMS). Late stent thrombosis after Cypher and Taxus drugeluting stent (DES) placement still has emerged as a major concern (Finn et al., 2007). The process involves a complex cascade of reactions that result in luminal narrowing through a combination of neointimal hyperplasia and constrictive
remodelling. The limitation of DES is the lack of reendothelialization related to inhibition of endothelial cell proliferation and migration since drug is delivered to the luminal surface (Finn et al., 2007; Seedial et al., 2013). 3Hydroxy-3-methyl-glutaryl-CoA reductase inhibitors, statins, are potent cholesterol-lowering drugs. Recently, in vivo and in vitro studies have demonstrated that, the drugs have additional vasculo-protective effects independent of cholesterol lowering: statins improve endothelial function, reduce vascular inflammation, decrease platelet aggregation and enhance endothelial processes involved in angiogenesis (Undas et al., 2005; Fadini et al., 2010). Moreover, statins
Corresponding author: e-mail: [email protected] These authors contributed equally to this work. Abbreviations: CMECs, cardiac microvascular endothelial cells; DES, drug-eluting stent; DHE, staining dihydroethidine; eNOS, endothelial nitric oxide synthase; FoxO, the ForkheadBox class O; mTOR, mammalian target of rapamycin; MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide]assay; NO, nitric oxide; p70S6K, p70 S6 kinase; PI3Ks, phosphoinositide 3-kinases; ROS, reactive oxygen species; WST-8, [2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium, monosodium salt] viability assay
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promote angiogenic processes, including CMECs proliferation and migration (Weis et al., 2002; Katsumoto et al., 2005) and exert direct beneficial effects on the endothelium in part through an increase in nitric oxide (NO) production (Brouet et al., 2001). However, the molecular mechanism underlying these serum lipid-independent effects of statins in CMECs is not well understood yet. It is well established that activation of phosphatidylinositol 3-kinase (PI3K) plays an important role in promoting cell survival and proliferation in numerous cell systems (Ruckerl et al., 2012; Thomas et al., 2013). And also as an essential role in angiogenesis (Yoshioka et al., 2012). In CMECs, it has been shown that the majority of growth factor-induced responses are mediated by the activation of the PI3K/Akt signalling cascade (Somanath et al., 2006) and statins could rapidly induce the activation of Akt in these cells (Brouet et al., 2001). Simvastatin has been shown to reduces venous stenosis formation in amurine hemodialysis vascular access model (Janardhanan et al., 2013). We have directed our attention to the molecular mechanisms by which simvastatin induces proliferation and migration in CMECs. We found that CMECs proliferation, migration and survival were enhanced by simvastatin, and this effect was mediated by PI3K/Akt-induced mammalian target of rapamycin (mTOR)/p70 S6 kinase (p70 S6K) and mTOR/FoxO3a activation in rat CMECs. Materials and methods
Animals Twenty male SD (Sprague–Dawley) rats (100–150 g) were purchased from the Experimental Animal Center of the Fourth Military Medical University. All animal procedures were performed in accordance with protocols approved by the Fourth Military Medical University Animal Research Committee and International Research Animal Care Guidelines.
Isolation, cultivation and identification of CMECs Briefly, CMECs were enzymatically released from left ventricular tissue of adult male SD rats using 0.2% collagenase type II (Worthington Biochemical Corp., Lakewood, NJ, USA) in sterile Hank’s buffer solution (HBSS) (Invitrogen, LuBio Science, Lucerne, Switzerland) for 20 min at 378C followed by 0.25% trypsin (Invitrogen) for 20 min at 378C in a shaking water bath according to the method developed by Nishida et al. (1993) with minor modifications. CMECs reached 80% confluence within 3–4 days in DMEM (Amimed, BioConcept, Allschwil, Switzerland) with 20% new-born calf serum (NCS) (Invitrogen, USA) and antibiotics. No additional growth factors were added to the growth medium. CMEC were then used in 600
the second passage and starved for 1 day in DMEM with 1% NCS before pharmacologic treatments. Cultured CMECs were identified by morphological and functional characteristics (Wei et al., 2010) with microscopy and Dil-ac-LDL intake assay (Molecular Probes, Eugene, OR, USA). Experiments were performed on cells at passage three to five from primary culture.
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide] assay CMECs were plated overnight in 96-well tissue culture plastic dishes at 1 104 cells/mL. Cells were treated with (1) rapamycin (Calbiochem, San Diego, CA, USA) at 0, 102, 101, 100, 101 nM or (2) rapamycin (100 nM) in the absence or the presence of simvastatin (Sigma–Aldrich, USA) at 0, 102, 101, 100, 101 mM for an additional 24 h. Cells from each group were harvested and 100 mL MTT (Sigma– Aldrich) was added into each well and incubated for 4 h. At the end of this treatment, the incubation medium was removed and formazan crystals were dissolved in 100 mL DMSO. MTTreduction was quantified by measuring the light absorbance at 490 nm.
WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] viability assay CMECs were plated into 96-well plates until 80% confluence then starved with DMEM 0.5% NCS for 24 h and treated in line with the MTT assay. CMECs were incubated for 2 h at 378C with WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] (CCK-8) (Dojindo, Labforce, Nunningen, Switzerland) while they released orange formazan into the medium and absorption was measured using a Safire multiwell-plate reader (Tecan). The WST-8 assay does not include washing steps and therefore is more suitable for stressed CMECs that tend to detach and get lost during the assay.
Cell migration assay For transwell migration assays, the cells were placed into the upper chamber on uncoated membranes (24-well insert; pore size 8 mm; BD Biosciences, California, USA). The cells were incubated for 24 h, and cells that did not migrate through the pores were removed using a cotton swab. The cells on the lower surface of the membrane were stained with gentian violet for 10 min and counted. Migration was assessed by counting cells in five microscopic fields per well at 400 magnification. The data are representative of triplicate experiments. Cell Biol Int 38 (2014) 599–609 ß 2013 International Federation for Cell Biology
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Assessment of apoptosis of CMECs
NO measurement
Following pretreatment for 24 h, CMECs were fixed, stained with Hochest 33258 (Sigma–Aldrich) and viewed by fluorescence microscopy (Olympus, Tokyo, Japan). Cells were designated as apoptosis with highly condensed, brightly staining nuclei and non-apoptosis with light blue staining. The apoptotic index was defined as the ratio of the apoptotic cell number to total cell number. Caspase-3 activity was determined by the Caspase-Activity Assay Kit (Chemicon, Billerica, USA).
The amount of NO production released by CMECs was determined by measuring the concentration of nitrite, a metabolite of NO, with a modified Griess reaction method (Nanjing Jiancheng Institute of Biological Engineering). Briefly, CMECs were seeded at 6 105 cells/well on a six-well plate one day before the treatment. Adherent cells were pretreated with rapamycin and/or LY294002 for 1 h and then with simvastatin for a further 24 h. Finally, supernatant was collected and mixed with an equal volume of modified Griess reagent for the colorimetric assay. After 10 min incubation at room temperature, the concentration of the resultant chromophore was measured spectrophotometrically at 550 nm after enzymatic conversion of the supernatant nitrate to nitrite by nitrate reductase. The nitrite concentration in the samples was calculated from nitrite standard curves made from sodium nitrite using the same culture medium.
Western blot analysis To determine the role of PI3K pathway, in addition to previous groups, we have added a group of rapamycin (100 nM) in the absence or the presence of simvastatin (100 mM) and LY294002 (Calbiochem; the inhibitor of PI3K) at 50 mM. Cells from each group were cultured and harvested at the indicated time. Cells were washed three times with cold PBS, and scraped off using an ice-cold lysis buffer. Proteins were prepared and separated on 10% SDS–PAGE gels before being transferred electrophoretically to nitrocellulose membranes. After blocking with 5% (w/v) non-fat dried skimmed milk powder for 1 h, the membranes were immunoblotted with the appropriate primary antibody at 48C overnight. The membranes were washed and further incubated with the secondary antibody at 378C for 60 min. Blots were visualized using an ECL (enhanced chemiluminescence system, Amersham). The following primary antibodies were used: Akt, phosphor-Akt (Ser73), FoxO3a, phospho-FoxO3a (Thr32), p70S 6K, phospho-p70S 6K (Thr389). Anti-bodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
Real-time reverse transcriptase PCR Real-time PCR assays were used on the samples to measure the expression of eNOS (QT0010075) and NOS2 (NM 010927). For gene analysed, reactions involved 1 mL Qiagen Quanti Tect Primer Assay added to 5 mL Power SyBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) and diluted with 2 mL molecular grade water. A final volume of 8 mL was dispensed into each well and 2 mL diluted cDNA (160 ng/reaction) was added. Each sample was tested in triplicate for each gene, and PCR reactions were performed using ABI Prism 7900 real-time PCR equipment. The thermal profile consists of 958C for 15 min, then 40 cycles of 948C for 15 s, 558C for 30 s and 728C for 30 s. This was plotted as a melting curve. The comparison between samples was performed using GAPDH and RPL32 as internal standards. REST MCS software was utilized for the calculation of the relative differences between the test groups. Cell Biol Int 38 (2014) 599–609 ß 2013 International Federation for Cell Biology
Quantification of ROS production in CMECs ROS production, an index of oxidative stress, in viable CMECs was measured by lucigenin-enhanced chemiluminescence assay adapted from the method (Schuhmacher et al., 2010). ROS production was expressed as relative light units (RLU) per second per million cells (RLU/s/million cells). Dihydroethidine (DHE; Beyotime, China) staining was used to detect the in situ formation of superoxide according to the oxidative fluorescent microtopography, as described recently (Zuo et al., 2011). DHE staining was visualized under a confocal microscope (Olympus). Images of cells were analysed with Image-Pro Plus software version 6.0. The mean fluorescence intensity of each cell was calculated, and the total cell emission signals per field were averaged for data analysis.
Statistical analysis All values were presented as means SD of n independent experiments. Most data (except Western blot density) were compared by ANOVA followed by Bonferroni correction for post hoc analysis. P < 0.05 was considered statistically significant. GraphPad Prism software version 5.0 (GraphPad Software) was used for the calculations. Results
Identification of CMECs The cells had ‘flagstone’ morphology (Figure 1A-a), and tested positive with the Dil-ac-LDL intake assay (Figure 1Ab), both indicating that the cultured cells were CMECs. 601
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Figure 1 Characterization of CMECs and to detected the proliferation and migration in rapamycin-induced CMECs. (A-a) CMECs monolayer presents cobble stone appearance by phase-contrast microscopy (scale bar: 40 mm); (A-b) Uptake of Dil-Ac-LDL by immunofluorescence (red, Dil-Ac-LDL; blue, DAPI, scale bar: 50 mm); CMECs were exposed to rapamycin for 24 h and proliferation was measured by the MTT-assay (B) and WST-8 assay (C). Representative image (D) of membranes where the lower well cell medium contained different concentration of rapamycin (scale bar: 550 mm); (E) Quantity of migration number of CMECs detected by transwell assays. P < 0.05, #P < 0.01 and &P < 0.001 versus control. Each column represents the mean SD of six different experiments. Abbreviations: Con, control; NS, no significant.
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Rapamycin suppresses CMECs proliferation and migration in a dose-dependent manner CMECs proliferation is a crucial process for angiogenesis. The MTT assay was used to investigate whether rapamycin is capable of restricting CMECs proliferation. Treated with the increasing concentrations of rapamycin for 24 h, the viability of CMECs was decreased in a dose-dependent manner (Figure 1B). Compared to the MTT assay, the WST-8 assay showed similar results in CMECs, with a significant reduction at 100 and 101 nM of rapamycin, respectively (Figure 1C). Migration assays were done with the transwell system. Consistent with its effects on proliferation, rapamycin also clearly suppressed CMECs migration (Figures 1D and 1E). Based on the results, we chose a concentration 100 nM for further study.
Protection of simvastatin in CMECs
the expression of caspase-3 activity and found that, simvastatin statistically decreased the caspase-3 expression in rapamycin-induced CMECs (Figure 2G). These results show that simvastatin had an anti-apoptotic effect on rapamycin-induced CMECs.
Simvastatin induces p70 S6K, FoxO3a phosphorylation via PI3K/Akt pathways in CMECs
To investigate whether simvastatin is capable of promoting CMEC proliferation, CMECs were then treated with vehicle or simvastatin (100 mM) only as control. After pretreated with rapamycin (100 nM), the cell viability in CMECs treated with different concentrations of simvastatin was significantly higher than that in rapamycin group by MTT assay (Figure 2A), especially treated with simvastatin at the concentrations 101 or 100, 101 mM. However, there was no significant difference between 100 and 101 mM simvastatin group. The WST-8 assay results (Figure 2B) were similar with MTTassay. The cell migration assays showed that supplement of simvastatin could improve the number of migrated cells into the lower chamber after pretreated with rapamycin (Figures 2C and 2D). Both of these data indicated that simvastatin at 100 mM significantly enhanced the viability and migration of CMECs after pretreated with rapamycin. However, there was no further effect of simvastatin administration at higher concentrations of 101 mM on the viability and migration of CMECs after pretreated with rapamycin.
In CMECs, the majority of growth factor-induced responses are mediated by the activation of PI3K/Akt signalling pathway (Somanath et al., 2006). These results prompted us to further investigate the mechanisms involved in the improvement of simvastatin after rapamycin-induced injury. We analysed whether simvastatin activates this pathway in CMECs. Cells were stimulated with 100 mM simvastatin for 24 h and cell lysates were prepared and subjected to Western blot analysis. Addition of simvastatin resulted in phosphorylation of Akt (Figures 3A and 3B). We tested the effect of simvastatin on the phosphorylation of a PI3K/Akt downstream target, p70 S6K. p70 S6K helps regulate the translation machinery and is controlled by mTOR, an immediate downstream effector of Akt (Miriuka et al., 2006). Simvastatin induced the phosphorylation of p70 S6K (Figures 3A and 3C), thereby leading to enzyme activation. Pretreatment of cells with LY294002, a specific PI3K inhibitor, abolished the phosphorylation of Akt and p70 S6K, whereas pretreatment with rapamycin, a specific inhibitor of mTOR, inhibited only p70 S6K activation (Figures 3A–3C). Thus simvastatin activates PI3K/Akt/mTOR/p70 S6K signalling in a sequential manner in CMECs. The Forkhead Box, class O (FoxO) family of Forkhead transcription factors, is a regulator of oxidative stress (Salih and Brunet, 2008). These transcription factor are also downstream targets of mTOR (Tesio et al., 2011) and are inactivated by Akt-dependent phosphorylation of serine/ threonine residues (Birkenkamp and Coffer, 2003). We investigated whether simvastatin phosphorylates and thereby inactivates the forkhead transcription factors. Simvastatin also phosphorylated FoxO3a with the same kinetics as for Akt and p70 S6K (Figures 3A and 3D).
Simvastatin prevented rapamycin-induced apoptosis of CMECs
Simvastatin promotes the secretion of NO in CMECs via a PI3K/Akt dependent mechanism
To investigate the role of simvastatin on rapamycin-induced apoptosis in CMECs, Hochest 33258 staining assay was performed. Rapamycin induced a significant increase in Hochest 33258 staining positive cells as brightly stained nuclei. Compared with cells cultured in rapamycin, each simvastatin treated group showed a significant decrease in apoptotic cells (Figures 2E and 2F), especially in the concentrations of simvastatin at 100 mM. We also detected
Rapamycin (100 nM) significantly decreased NO secretion in comparison with the control group (21.7 2.9 mmol/L vs. 36.3 5.8 mmol/L, P < 0.01; Figure 4A). However, pretreatment with simvastatin (100 mM) enhanced the NO secretion both in basal (46.7 7.3 mmol/L vs. 36.3 5.8 mmol/L, P < 0.01) and in rapamycin-induced groups (35.1 3.6 mmol/L vs. 21.7 2.9 mmol/L, P < 0.01). Real time PCR analysis showed that rapamycin downregulated the
Simvastatin significantly improves the proliferation and migration in CMECs
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Figure 2 Effect of simvastatin on CMECs’ proliferation, migration and apoptosis. CMECs were treated with rapamycin in the absence or the presence of simvastatin. Viable cell numbers were monitored by MTT assay (A) and WST assay (B); Representative image (C, scale bar: 550 mm) and quantity (D) of migration detected by transwell assay. CMECs were stained with Hochest 33258 and observed by fluorescence microscopy. Representative image (E, scale bar: 50 mm) and quantity (F) of apoptosis by simvastatin treated. (G) Expression of caspase-3 activity assay was quantified. Each column represents the mean SD of six different experiments. P < 0.05, versus control, #P < 0.05 versus rapamycin (100 nM) alone group, &P < 0.05 versus simvastatin at 100 mM group. Abbreviations: Con, control; S, simvastatin; R, rapamycin; NS, no significant.
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Figure 3 Effect of simvastatin induces phosphorylation of Akt, p70 S6K and FoxO3a in a PI3K-dependent manner. Western blotting was performed to analyse the phosphorylation of Akt, p70 S6K and FoxO3a. (A) Representative blot. Quantitative densitometry data of pAkt (B), p-p70 S6K (C) and pFoxO3a (D) expression of CMECs in different groups. As a loading control, the membranes were stripped and reprobed with antibodies for the total level of each protein. Each column represents the mean SD of six different experiments. P < 0.01, versus control, #P < 0.05 versus rapamycin (100 nM) alone group, &P < 0.01 versus rapamycin (100 nM) and simvastatin (100 mM) and LY294002 (50 mM) group. Abbreviations: Con, control; S, simvastatin; R, rapamycin; LY, LY294002, Akt inhibitor.
expression of eNOS mRNAs in CMECs (P < 0.05), whereas pretreatment with simvastatin upregulated the expression (Figure 4B, P < 0.05). The data indicated that simvastatin pretreatment increased NO release in both basal and rapamycin-induced groups by increasing the expression of eNOS. Both of these effects, including NO secretion and eNOS expression, were attenuated with pretreatment with LY294002.
cence assay (Figure 5C). Both suggested that exposure of CMECs to rapamycin promoted intracellular ROS levels, which was attenuated by simvastatin. However, the inhibitory effect of simvastatin on rapamycin-induced ROS production was abolished by LY294002. Thus simvastatin might decrease rapamycin-induced ROS accumulation in CMECs through PI3K/Akt signalling pathway. Discussion
Simvastatin decreases rapamycin-induced ROS production in CMECs To determine the mechanism of action behind the beneficial effect of simvastatin on CMECs, ROS production was measured by two independent approaches: DHE staining (Figures 5A and 5B) and lucigenin-enhanced chemiluminesCell Biol Int 38 (2014) 599–609 ß 2013 International Federation for Cell Biology
Without exception, all interventions designed to treat atherosclerotic occlusive disease are complicated by restenosis (Seedial et al., 2013). Despite many advances in the fields of vascular biology, pharmacology and bioengineering, restenosis remains a significant problem (Clowes et al., 1983a, b). One of the reasons causing restenosis is 605
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Figure 4 Effects of simvastatin on NO release and eNOS expression in rapamycin-induced CMECs. Simvastatin significantly increases NO secretion (A) both in basal and in rapamycin-induced groups. (B) eNOS mRNA data. This improvement (including NO secretion and eNOS expression) by simvastatin could attenuate with pretreatment with LY294002. Each column represents the mean SD of six different experiments. P < 0.05, versus control, #P < 0.05 versus rapamycin alone group, &P < 0.01 versus rapamycin (100 nM) and simvastatin (100 mM) and LY294002 (50 mM) group. Abbreviations: Con, control; S, simvastatin; R, rapamycin; LY, LY294002, Akt inhibitor.
related to inhibition of endothelial cell proliferation and migration since drug is delivered to the luminal surface from DES (Finn et al., 2007; Seedial et al., 2013). All forms of angiogenesis may share certain basic features, including
CMECs proliferation and migration, etc. (Carmeliet, 2003) and are regulated by many pro-angiogenic growth factors (Suri et al., 1996). Other than these growth factors (such as VEGF etc.), simvastatin (Shen et al., 2011) and rosuvastatin
Figure 5 Effects of simvastatin on rapamycin-induced oxidative stress in CMECs. (A) Representative diagram showing DHE staining of CMECs (red, DHE; blue, DAPI). (B) The average fluorescence intensity from five fields was summarized. (C) Superoxide generation of CMECs in different groups. Each column represents the mean SD of six different experiments. Scale bar: 40 mm. P < 0.01, versus control, #P < 0.05 versus rapamycin (100 nM) alone group, &P < 0.05 versus rapamycin (100 nM) and simvastatin (100 mM) and LY294002 (50 mM) group. Abbreviations: Con, control; S, simvastatin; R, rapamycin; LY, LY294002, Akt inhibitor; DHE, dihydroethidine.
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(Zhou et al., 2013) have an angiogenic effect on CMECs. CMECs proliferation is a crucial process for angiogenesis. Cerivastatin and pitavastatin stimulate CMECs proliferation (Weis et al., 2002; Katsumoto et al., 2005). Statins have an anti-apoptotic effect on endothelial progenitor cells (Ruckerl et al., 2012). Therefore, in investigating whether simvastatin promotes CMECs proliferation, migration and survival after pretreated with rapamycin, we found that low concentration (100 mM) of simvastatin could significantly improve proliferation, migration and apoptosis in CMECs, whereas higher concentrations (101 mM) offered no further improvement consistent Katsumoto et al. (2005). The results confirmed that, at therapeutic doses, statins promoted proliferation, migration and survival of CMECs. Phosphorylation of Akt targets may contribute to CMECs functioning, including survival, growth and migration (Somanath et al., 2006). Activation of Akt promotes endothelial cell survival by inhibiting apoptosis (Gerber et al., 1998; Dimmeler et al., 2001), stimulating endothelial NO synthesis (Dimmeler et al., 1999). mTOR is an important downstream target of PI3K/Akt, and mTOR signalling is involved in the control of cell growth and proliferation (Sarbassov et al., 2005a). mTORC1 activation leads to phosphorylation of p70 S6K, and mTORC1 signalling is inhibited by rapamycin (Jacinto et al., 2004). mTORC1 is the downstream target of the Akt and the PI3K/Akt/mTOR/p70 S6K pathway assists in the control of cell growth and proliferation (Hay and Sonenberg, 2004; Sarbassov et al., 2005a). The role of the mTOR pathway involved in rapamycin-induced CMECs injury is not fully understood. Many studies (Jacinto et al., 2004; Sarbassov et al., 2005a; Zhang et al., 2005; Li et al., 2007) indicate that the mTOR signalling pathway is important in angiogenesis. To demonstrate that Akt is indeed an essential down-stream signalling event, Akt phosphorylation (indicative of Akt activity) was detected by Western blotting. Simvastatin also robustly activates the Akt/p70 S6K pathway in CMECs, and this activation is completely inhibited by LY294002. Whereas pretreatment with rapamycin, a specific inhibitor of mTOR, inhibits only p70 S6K activation. Because PI3K has many downstream targets that are essential for cell survival, it is possible that inhibition of PI3K leads to cell death and thereby accounts for the decrease in the CMECs’ proliferation, migration and survival. On the other hand, rapamycin inhibits both basal and simvastatin-induced p70 S6K activity, but the amount of suppression is smaller than that treated by PI3K inhibition. These results raise the possibility that simvastatin enhances angiogenesis via the PI3K/Akt/mTOR/ p70 S6K signalling pathway (Figure 6). Iantorno et al. (2007) found that PI3K/Akt signalling pathway is involved in production of NO in CMECs. NO is the most important vasodilator (Moncada and Higgs, 2006), and rapamycin can inhibit NO secretion in a dose-dependent Cell Biol Int 38 (2014) 599–609 ß 2013 International Federation for Cell Biology
Protection of simvastatin in CMECs
Figure 6 Proposed scheme for protection effect by simvastatin in CMECs. Simvastatin activates PI3K/Akt/mTOR pathways, which inactivate FoxO3a and p70 S6K by phosphorylation. FoxO3a, members of the FoxO (Forkhead box, class O) subfamily of forkhead transcription factors is a regulator of oxidative stress and p70S6K, a key translation initiation and elongation regulator, both of them can be phosphorylated by mTOR. Subsequently, FoxO3a inhibition by rapamycin and LY294002 causes increased ROS production. Rapamycin can inhibit NO secretion directly. Simvastatin increased levels of PI3K/Akt/mTOR phosphorylation, which improve the CMEC proliferative, migration and anti-apoptosis after rapamycin-induction.
manner (Miriuka et al., 2006). We found that simvastatin pretreatment increased eNOS activity, thus mediating the upregulation of NO in CMECs. We also showed that the effect of simvastatin is closely related to the PI3K/Akt activity. LY294002, a highly selective PI3K inhibitor, almost completely inhibited simvastatin-induced Akt phosphorylation in CMECs and markedly suppressed simvastatininduced CMECs secretion of NO. Pretreatment with rapamycin inhibited only the activation of mTOR downstream molecules and had little effect on NO secretion. These results suggest that the effect of PI3K activation of NO secretion in CMECs is mediated in part by the mTOR pathway, which could be the mechanism behind its favourable effects on neovascularization. The apoptosis of CMECs may be in part due to other molecules that are not related to PI3K/Akt/mTOR/p70 S6K signalling. FoxOs are a direct substrate of the protein kinase 607
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Protection of simvastatin in CMECs
Akt, a mammalian target of rapamycin inhibition (mTOR) target (Sarbassov et al., 2005b), which inactivates them by phosphorylation (Brunet et al., 1999). Activation of Akt signalling cascades mediated anti-apoptotic and pro-proliferative signals by causing FoxO3a phosphorylation, inactivation and consequent cytoplasmic entry (Yang et al., 2008) and FoxO3a negatively regulates an anti-apoptotic microRNA (Wang and Li, 2010). Since phosphorylation determines the nuclear/cytoplasmic location of FoxO3a activity, we also tested whether simvastatin could induce the phosphorylation of FoxO3a; it activated the mTOR/FoxO3a pathway in CMECs, which was inhibited by both LY294002 and rapamycin. c-Met activation induced mTOR signalling, downregulates FoxO3a levels and ultimately increases ROS production (Tesio et al., 2011). Low levels of ROS are indispensable in many biochemical processes, including intracellular signalling, defence against microorganisms, and cell functioning (Go and Jones, 2011). In contrast, high dose and/or inadequate removal of ROS results in oxidative stress, which has been implicated in severe pathophysiological consequences (Fattman et al., 2003). Through the detection of ROS production, we found that simvastatin decreased rapamycin-induced ROS production, but did not markedly affect basal ROS production. This improvement can be abrogated by PI3K inhibition. Our data suggest mTOR/ FoxO3a/ROS signalling cascade, which decreases ROS levels, may be a part of beneficial effects of simvastatin. After pretreatment with LY294002, the significant increase of ROS production may be in part due to other molecules that are not only related to PI3K/Akt/mTOR/FoxO3a signalling (Figure 6). In summary, simvastatin has a biphasic effect on CMEC proliferation, migration and anti-apoptosis. Low concentrations of simvastatin can induce proliferation, migration and anti-apoptosis via PI3K/Akt/mTOR/p70 S6K and mTOR/FoxO3 signalling pathways, and then exert a beneficial effect by adjustment of NO and ROS production in CMECs. The possibility is raised that the Akt/mTOR pathway is important in some of the pleiotropic effects of statins.
Acknowledgement and funding The National Natural Science Foundation of China (no. E011002) supported this work.
Author contribution Qiao Pan, Xiaobo Xie, Haichang Wang, Yan Guo conceived and designed the experiments; Qiao Pan, Xiaobo Xie, Haichang Wang, Yan Guo performed the experiments; Qiao Pan, Xiaobo Xie,Yan Guo analysed the data and
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