CVR-2011-1235R2
Supplemental Material and Methods
1. Cell death detection, DNA fragmentation, and cell proliferation assay
Cell death was measured by cytoplasmic-associated DNA fragments using Cell Death Detection ELISA
(Roche, Mannheim, Germany). Apoptotic DNA-Ladder was detected using Apoptotic DNA Ladder Kit
(Roche). Cell growth was evaluated using Cell Proliferation ELISA, BrdU (colorimetric) (Roche). These
assays were performed according to the manufacturer’s instructions.
2. Real-time quantitative RT-PCR analysis
Transcript levels of target genes were identified by reverse transcription and quantitative PCR using High
Capacity RNA-to-cDNA Kit (Applied Biosystems, NY, USA) , TaqMan PCR Master Mix (Applied
Biosystems,), and FAM-labeled TaqMan probes (Assays-on-Demand, Applied Biosystems) for human
preproET-1 (Applied Biosystems, Hs00174961_m1) , HIF-1α (Applied Biosystems, Hs00153153_m1),
low-density
lipoprotein
glyceraldehyde-3-phosphate
receptor
(LDL-R)
dehydrogenase
(Applied
(GAPDH)
Biosystems,
(Applied
Hs00181192_m1),
Biosystems,
and
Hs00266705_g1).
Expression data were normalized for GAPDH levels.
3. Western blot assay
Whole-cell lysates were subjected to Western blot analyses (10% SDS-PAGE or 5–20% gradient
SDS-PAGE; 20 μg protein per lane) using anti-HIF-1α (sc-10790, Santa Cruz Biotechnology, CA, USA)
at 1 : 5000 dilution, anti-HIF-1β (sc-5580, Santa Cruz Biotechnology) at 1 : 3000 dilution, anti-VHL
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CVR-2011-1235R2
(sc-17780, Santa Cruz Biotechnology) at 1 : 1000 dilution, anti-HA antibody (Clontech, Mountain View,
CA, USA) at 1 : 5000 dilution, and anti-β-actin (Sigma) at 1 : 10000 dilution. Subsequently, the
membranes were incubated with a secondary antibody conjugated to horseradish peroxidase (GE
Healthcare, Little Chalfont, UK) at 1 : 10000 dilution. The proteins were visualized and quantified using a
chemiluminescence method (ECL Plus Western Blotting Detection System, GE Healthcare).
4. Chromatin immunoprecipitation (ChIP) assay
VSMC (2 × 106 cells) were cross-linked using 1% formaldehyde for 10 min at 37°C. Cross-linking was
stopped by adding glycine (final concentration, 150 mmol/L) to the medium and incubating for 5 min at
room temperature. After suspension with ice-cold phosphate buffered saline containing protease
inhibitors and centrifugation, the cell pellets were subsequently resuspended in 900 μL lysis buffer (0.1%
SDS, 300 mmol/L NaCl, 1 mmol/L EDTA, 0.5 mmol/L EGTA, 0.5% N-lauroylsarcosine, and 10 mmol/L
Tris–HCl [pH 8.0]) and incubated on ice for 10 min. The lysates were sonicated using a Bioruptor
Ultrasonicator UCD-250 (Cosmo-Bio, Tokyo, Japan) and the chromatin in each lysate was sheared to an
average size of 500 bp. After addition of 90 μL of 10% Triton X-100 and centrifugation at 12,000 xg for
20 min, the supernatants were collected and incubated with rotation at 4°C overnight in the presence of
Dynabeads M-280 for anti-rabbit IgG (Invitrogen), which were pre-bound with 4 μg anti-HIF-1α antibody
or rabbit IgG (Santa Cruz Biotechnology). After incubation, the beads were washed once with low-salt
wash buffer (0.1% SDS, 150 mmol/L NaCl, 1% Triton X-100, 2 mmol/L EDTA, and 20 mmol/L Tris,
[pH 8.0]), twice with high-salt wash buffer (0.1% SDS, 400 mmol/L NaCl, 1% Triton X-100, 2 mmol/L
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CVR-2011-1235R2
EDTA, and 20 mmol/L Tris [pH 8.0]), five times with LiCl wash buffer (0.5 M LiCl, 1% Nonidet P-40,
0.7% deoxycholate, 1 mmol/L EDTA, and 50 mmol/L HEPES [pH 7.6]), and finally once with
Tris–EDTA buffer. After centrifugation, immune complexes were resuspended in 200 μL elution buffer
(1% SDS, 50 mmol/L Tris–HCl [pH 8.0], and 10 mmol/L EDTA) and incubated for 15 min at 65°C.
Cross-linking of immune complexes was reversed in 200 mmol/L NaCl at 65°C overnight, after which
complexes were digested with proteinase K at 45°C for 1 h. Genomic DNA fragments were recovered by
phenol–chloroform extraction, ethanol precipitation, and resuspension in sterile H2O. Human genomic
sequences containing HRE from −204 bp to +173 bp relative to the transcription start site of the ET-1
gene were amplified using the following primers: ET-1 forward, 5′-CTGCCCCGAATTGTCAGACG-3′;
ET-1 reverse, 5′-CAAAGCGATCCTTCAGCCC-3′.
Supplemental Discussion
Previous studies reported that statin affects the function of the mammalian target of rapamycin1, an
important factor in HIF-1α synthesis2. However, there was no significant effect of fluvastatin on HIF-1α
protein level in the presence of MG-132 or CoCl2, as shown in Figures 4A and 5. Therefore, the main
inhibitory effect of statin on HIF-1α protein level is the acceleration of protein degradation.
The relationships between HIF-1 and other transcription factors, including NF-κB-dependent
HIF-1α transcription and the interaction between HIF-1 and AP-1, have been investigated3,4. Several
studies showed the inhibitory effect of statin on these transcription factors5,6. The effect of statin on these
transcription factors may interfere with or enhance HIF-1-dependent ET-1 regulation. However, the
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CVR-2011-1235R2
pattern of inhibition of HIF-1 protein by fluvastatin was similar to that of hypoxia-dependent ET-1
expression and there was no significant effect on HIF-1α mRNA level in our study. Therefore, the
crosstalk between HIF-1 and these transcriptional factors may not have a great influence.
Oxidative stress reportedly affects HIF-1 function: Sirtuin 3 attenuates HIF-1α accumulation in
hypoxia by suppression of mitochondrial reactive oxygen species in carcinoma cell lines7. Previous
investigations reported that statins inhibited cellular oxidative stress, including inhibition of nicotinamide
adenine dinucleotide phosphate hydrogen (NADPH) oxidase 1 expression and changes in superoxide
dismutase (SOD) levels as pleiotropic effects8,9, and it is possible that this involves the attenuation of
hypoxia-dependent ET-1 induction. On the other hand, fluvastatin has the unique structure of a
fluorophenyl-substituted indole ring that exerts an additional antioxidant effect10, but other statins, such
as pravastatin and simvastatin, do not have this structure. Therefore, there may be a difference between
fluvastatin and other statins in the effect on cellular oxidative stress. Nevertheless, the inhibitory effect on
HIF-1-dependent ET-1 expression was similar among the three types of statin (fluvastatin, pravastatin,
and simvastatin), as shown in Figure 2B, Supplemental Figure 3A, and 3C. Based on these findings, the
influence of statin on cellular oxidative stress may not involve hypoxia-induced ET-1 expression.
Previous studies in vascular endothelial cells have revealed that statins inhibit preproET-1
mRNA expression and preproET-1 promoter activity using 0.65 kb, 1.5 kb, and 5.2 kb of the preproET-1
5’ flanking region under normoxic conditions11. These studies also stated that high concentrations of
statins are required for these inhibitory effects: ≥2 μM of simvastatin or ≥5 μM of atorvastatin with more
than 12 h of incubation in bovine aortic endothelial cells12, and ≥1 μM of fluvastatin with more than 6 h
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CVR-2011-1235R2
of incubation in human umbilical vein endothelial cells
13
. Our findings in vascular smooth muscle cells
under normoxic conditions almost agree with these previous findings, although there are minor
differences in time course and statin concentrations. Treatment with lower concentration (100 nM) of
fluvastatin for >8 h affected preproET-1 mRNA expression, as shown in Figure 2C. Moreover, the
previous studies did not analyze hypoxia-induced preproET-1 gene expression. Our study is the first
report, as per our knowledge, of the inhibitory effects of fluvastatin on preproET-1 expression under both
normoxic and hypoxic conditions.
Supplementary References
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Wagner RJ, Martin KA, Powell RJ, Rzucidlo EM. Lovastatin induces VSMC differentiation through
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2.
Nakamura H, Makino Y, Okamoto K, Poellinger L, Ohnuma K, Morimoto C, et al. TCR engagement
increases hypoxia-inducible factor-1 alpha protein synthesis via rapamycin-sensitive pathway under
hypoxic conditions in human peripheral T cells. J Immunol 2005;174:7592-9.
3.
Diebold I, Djordjevic T, Hess J, Gorlach A. Rac-1 promotes pulmonary artery smooth muscle cell
proliferation by upregulation of plasminogen activator inhibitor-1: role of NFkappaB-dependent
hypoxia-inducible factor-1alpha transcription. Thromb Haemost 2008;100:1021-8.
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Laderoute KR. The interaction between HIF-1 and AP-1 transcription factors in response to low
oxygen. Semin Cell Dev Biol 2005;16:502-13.
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Hattori Y, Matsumura M, Kasai K. Vascular smooth muscle cell activation by C-reactive protein.
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Cardiovasc Res 2003;58:186-95.
6.
Viedt C, Shen W, Fei J, Kamimura M, Hansch GM, Katus HA, et al. HMG-CoA reductase inhibition
reduces the proinflammatory activation of human vascular smooth muscle cells by the terminal
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Bell EL, Emerling BM, Ricoult SJ, Guarente L. SirT3 suppresses hypoxia inducible factor 1alpha
and tumor growth by inhibiting mitochondrial ROS production. Oncogene 2011;30:2986-96.
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Briones AM, Rodriguez-Criado N, Hernanz R, Garcia-Redondo AB, Rodrigues-Diez RR, Alonso MJ,
et al. Atorvastatin prevents angiotensin II-induced vascular remodeling and oxidative stress.
Hypertension 2009;54:142-9.
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Verreth W, De Keyzer D, Davey PC, Geeraert B, Mertens A, Herregods MC, et al. Rosuvastatin
restores superoxide dismutase expression and inhibits accumulation of oxidized LDL in the aortic
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10. Blum CB. Comparison of properties of four inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A
reductase. Am J Cardiol 1994;73:3D-11D.
11. Hernandez-Perera O, Perez-Sala D, Soria E, Lamas S. Involvement of Rho GTPases in the
transcriptional inhibition of preproendothelin-1 gene expression by simvastatin in vascular
endothelial cells. Circ Res 2000;87:616-22.
12. Hernandez-Perera O, Perez-Sala D, Navarro-Antolin J, Sanchez-Pascuala R, Hernandez G, Diaz C, et
al. Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin,
on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells. J
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Clin Invest 1998;101:2711-9.
13. Ozaki K, Yamamoto T, Ishibashi T, Matsubara T, Nishio M, Aizawa Y. Regulation of endothelial
nitric oxide synthase and endothelin-1 expression by fluvastatin in human vascular endothelial cells.
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