Induction of the Cytoprotective Enzyme Heme Oxygenase-1 by Statins Is Enhanced in Vascular Endothelium Exposed to Laminar Shear Stress and Impaired by Disturbed Flow
2009; Elsevier BV; Volume: 284; Issue: 28 Linguagem: Inglês
10.1074/jbc.m109.009886
ISSN1083-351X
AutoresFaisal Ali, Mustafa Zakkar, Kersti Karu, Elaine A. Lidington, Shahir Hamdulay, Joseph J. Boyle, Mire Zloh, Andrea S. Bauer, Dorian O. Haskard, Paul C. Evans, Justin C. Mason,
Tópico(s)Inflammasome and immune disorders
ResumoIn addition to cholesterol-lowering properties, statins exhibit lipid-independent immunomodulatory, anti-inflammatory actions. However, high concentrations are typically required to induce these effects in vitro, raising questions concerning therapeutic relevance. We present evidence that endothelial cell sensitivity to statins depends upon shear stress. Using heme oxygenase-1 expression as a model, we demonstrate differential heme oxygenase-1 induction by atorvastatin in atheroresistant compared with atheroprone sites of the murine aorta. In vitro, exposure of human endothelial cells to laminar shear stress significantly reduced the statin concentration required to induce heme oxygenase-1 and protect against H2O2-mediated injury. Synergy was observed between laminar shear stress and atorvastatin, resulting in optimal expression of heme oxygenase-1 and resistance to oxidative stress, a response inhibited by heme oxygenase-1 small interfering RNA. Moreover, treatment of laminar shear stress-exposed endothelial cells resulted in a significant fall in intracellular cholesterol. Mechanistically, synergy required Akt phosphorylation, activation of Kruppel-like factor 2, NF-E2-related factor-2 (Nrf2), increased nitric-oxide synthase activity, and enhanced HO-1 mRNA stability. In contrast, heme oxygenase-1 induction by atorvastatin in endothelial cells exposed to oscillatory flow was markedly attenuated. We have identified a novel relationship between laminar shear stress and statins, demonstrating that atorvastatin-mediated heme oxygenase-1-dependent antioxidant effects are laminar shear stress-dependent, proving the principle that biomechanical signaling contributes significantly to endothelial responsiveness to pharmacological agents. Our findings suggest statin pleiotropy may be suboptimal at disturbed flow atherosusceptible sites, emphasizing the need for more specific therapeutic agents, such as those targeting Kruppel-like factor 2 or Nrf2. In addition to cholesterol-lowering properties, statins exhibit lipid-independent immunomodulatory, anti-inflammatory actions. However, high concentrations are typically required to induce these effects in vitro, raising questions concerning therapeutic relevance. We present evidence that endothelial cell sensitivity to statins depends upon shear stress. Using heme oxygenase-1 expression as a model, we demonstrate differential heme oxygenase-1 induction by atorvastatin in atheroresistant compared with atheroprone sites of the murine aorta. In vitro, exposure of human endothelial cells to laminar shear stress significantly reduced the statin concentration required to induce heme oxygenase-1 and protect against H2O2-mediated injury. Synergy was observed between laminar shear stress and atorvastatin, resulting in optimal expression of heme oxygenase-1 and resistance to oxidative stress, a response inhibited by heme oxygenase-1 small interfering RNA. Moreover, treatment of laminar shear stress-exposed endothelial cells resulted in a significant fall in intracellular cholesterol. Mechanistically, synergy required Akt phosphorylation, activation of Kruppel-like factor 2, NF-E2-related factor-2 (Nrf2), increased nitric-oxide synthase activity, and enhanced HO-1 mRNA stability. In contrast, heme oxygenase-1 induction by atorvastatin in endothelial cells exposed to oscillatory flow was markedly attenuated. We have identified a novel relationship between laminar shear stress and statins, demonstrating that atorvastatin-mediated heme oxygenase-1-dependent antioxidant effects are laminar shear stress-dependent, proving the principle that biomechanical signaling contributes significantly to endothelial responsiveness to pharmacological agents. Our findings suggest statin pleiotropy may be suboptimal at disturbed flow atherosusceptible sites, emphasizing the need for more specific therapeutic agents, such as those targeting Kruppel-like factor 2 or Nrf2. The efficacy of 3-hydroxy-3-methylglutaryl-coenzyme A reductase antagonists (statins) in reducing low density lipoprotein cholesterol, cardiovascular morbidity, and mortality is widely recognized (1.Jain M.K. Ridker P.M. Nat. Rev. Drug Discov. 2005; 4: 977-987Crossref PubMed Scopus (704) Google Scholar). The observation that beneficial actions of statins on vascular function are detectable prior to any fall in serum cholesterol, extend to normocholesterolemic patients and exceed those of other lipid-lowering drugs despite comparable falls in total cholesterol (2.Landmesser U. Bahlmann F. Mueller M. Spiekermann S. Kirchhoff N. Schulz S. Manes C. Fischer D. de Groot K. Fliser D. Fauler G. März W. Drexler H. Circulation. 2005; 111: 2356-2363Crossref PubMed Scopus (413) Google Scholar, 3.Ridker P.M. Danielson E. Fonseca F.A. Genest J. Gotto Jr., A.M. Kastelein J.J. Koenig W. Libby P. Lorenzatti A.J. MacFadyen J.G. Nordestgaard B.G. Shepherd J. Willerson J.T. Glynn R.J. N. Engl. J. Med. 2008; 359: 2195-2207Crossref PubMed Scopus (5296) Google Scholar), suggest the existence of low density lipoprotein-cholesterol-independent effects (4.Mason J.C. Clin. Sci. 2003; 105: 251-266Crossref PubMed Scopus (106) Google Scholar, 5.Liao J.K. Laufs U. Annu. Rev. Pharmacol. Toxicol. 2005; 45: 89-118Crossref PubMed Scopus (1386) Google Scholar). Judging from in vitro studies, these may include immunomodulatory, anti-inflammatory, anti-adhesive, anti-thrombotic, and cytoprotective actions (6.Greenwood J. Mason J.C. Trends Immunol. 2007; 28: 88-98Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). However, the experimental work demonstrating these pleiotropic effects has predominantly used statin concentrations exceeding those achieved by therapeutic dosing, raising questions concerning clinical relevance (4.Mason J.C. Clin. Sci. 2003; 105: 251-266Crossref PubMed Scopus (106) Google Scholar). Heme oxygenase-1 (HO-1) 2The abbreviations used are: HO-1heme oxygenase-1ECendothelial cellsLSSlaminar shear stressNOnitric oxideeNOSendothelial nitric-oxide synthaseKLF2Kruppel-like factor 2Nrf2NF-E2-related factor-2MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidePI3Kphosphoinositide 3-kinasel-NAMENG-nitro-l-arginine methyl esterHUVEChuman umbilical vein endothelial cellsOFoscillatory flowDNdominant-negativeH2DCFdihydrodichlorofluoresceinSTAT3signal transducer and activator of transcription 3CM-H2DCFDA5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl estersiRNAsmall interfering RNARTreverse transcriptase. acts as the rate-limiting factor in the catabolism of heme into biliverdin, releasing free iron and carbon monoxide (CO). Biliverdin is subsequently converted to bilirubin by biliverdin reductase, whereas intracellular iron induces expression of heavy chain-ferritin and the opening of Fe2+ export channels (7.Ryter S.W. Alam J. Choi A.M. Physiol. Rev. 2006; 86: 583-650Crossref PubMed Scopus (1901) Google Scholar). The biologic activity of HO-1 represents an important adaptive response in cellular homeostasis, as revealed by widespread inflammation and persistent endothelial injury in human HO-1 deficiency (8.Yachie A. Niida Y. Wada T. Igarashi N. Kaneda H. Toma T. Ohta K. Kasahara Y. Koizumi S. J. Clin. Invest. 1999; 103: 129-135Crossref PubMed Scopus (1094) Google Scholar). heme oxygenase-1 endothelial cells laminar shear stress nitric oxide endothelial nitric-oxide synthase Kruppel-like factor 2 NF-E2-related factor-2 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide phosphoinositide 3-kinase NG-nitro-l-arginine methyl ester human umbilical vein endothelial cells oscillatory flow dominant-negative dihydrodichlorofluorescein signal transducer and activator of transcription 3 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester small interfering RNA reverse transcriptase. Expression of HO-1 in atherosclerotic lesions, and its ability to inhibit vascular smooth muscle cell proliferation, exert anti-inflammatory, antioxidant, and antithrombotic effects, suggests a protective role during atherogenesis (9.Wang L.J. Lee T.S. Lee F.Y. Pai R.C. Chau L.Y. Am. J. Pathol. 1998; 152: 711-720PubMed Google Scholar, 10.Stocker R. Perrella M.A. Circulation. 2006; 114: 2178-2189Crossref PubMed Scopus (199) Google Scholar). HMOX1 promoter polymorphisms affecting HO-1 expression may influence susceptibility to intimal hyperplasia and coronary artery disease, whereas a low serum bilirubin constitutes a cardiovascular risk factor (11.Exner M. Minar E. Wagner O. Schillinger M. Free Radic. Biol. Med. 2004; 37: 1097-1104Crossref PubMed Scopus (325) Google Scholar). Moreover, overexpression of HO-1 inhibited atherogenesis, whereas Hmox1−/− mice bred onto an ApoE−/− background developed more extensive and complex atherosclerotic plaques (12.Juan S.H. Lee T.S. Tseng K.W. Liou J.Y. Shyue S.K. Wu K.K. Chau L.Y. Circulation. 2001; 104: 1519-1525Crossref PubMed Scopus (302) Google Scholar, 13.Yet S.F. Layne M.D. Liu X. Chen Y.H. Ith B. Sibinga N.E. Perrella M.A. FASEB J. 2003; 17: 1759-1761Crossref PubMed Scopus (259) Google Scholar). Recent interest has focused on the therapeutic potential of HO-1 and its products, with probucol, statins, rapamycin, nitric oxide donors, and aspirin being shown to induce HO-1 (reviewed in Ref. 10.Stocker R. Perrella M.A. Circulation. 2006; 114: 2178-2189Crossref PubMed Scopus (199) Google Scholar). Indeed, induction of HO-1 may represent an important component of the vasculoprotective profile of statins, with simvastatin, atorvastatin, and rosuvastatin variously shown to increase HMOX1 promoter activity and mRNA levels, to induce enzyme activity and increase antioxidant capacity in human endothelial cells (EC) (14.Lee T.S. Chang C.C. Zhu Y. Shyy J.Y. Circulation. 2004; 110: 1296-1302Crossref PubMed Scopus (255) Google Scholar, 15.Dulak J. Loboda A. Jazwa A. Zagorska A. Dörler J. Alber H. Dichtl W. Weidinger F. Frick M. Jozkowicz A. Endothelium. 2005; 12: 233-241Crossref PubMed Scopus (66) Google Scholar, 16.Uchiyama T. Atsuta H. Utsugi T. Ohyama Y. Nakamura T. Nakai A. Nakata M. Maruyama I. Tomura H. Okajima F. Tomono S. Kawazu S. Nagai R. Kurarbayashi M. Atherosclerosis. 2006; 188: 265-273Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 17.Muchova L. Wong R.J. Hsu M. Morioka I. Vitek L. Zelenka J. Schröder H. Stevenson D.K. Can. J. Physiol. Pharmacol. 2007; 85: 800-810Crossref PubMed Scopus (60) Google Scholar, 18.Ali F. Hamdulay S.S. Kinderlerer A.R. Boyle J.J. Lidington E.A. Yamaguchi T. Soares M.P. Haskard D.O. Randi A.M. Mason J.C. J. Thromb. Haemost. 2007; 5: 2537-2546Crossref PubMed Scopus (79) Google Scholar). However, induction of HO-1 in vascular EC in vivo has not yet been demonstrated. Vascular endothelium exposed to unidirectional, pulsatile laminar shear stress (LSS) >10 dynes/cm2 is relatively protected against atherogenesis. LSS increases nitric oxide (NO) biosynthesis, prolongs EC survival, and generates an anticoagulant, anti-adhesive cell surface. In contrast, endothelium exposed to disturbed blood flow, with low shear reversing or oscillatory flow patterns, such as that located at arterial branch points and curvatures, is atheroprone. Thus endothelial cells exposed to disturbed blood flow exhibit reduced levels of endothelial nitric-oxide synthase (eNOS), increased apoptosis, oxidative stress, permeability to low density lipoprotein, and leukocyte adhesion (19.Berk B.C. Circulation. 2008; 117: 1082-1089Crossref PubMed Scopus (115) Google Scholar). The atheroprotective influence of unidirectional LSS and the overlap between these actions and those of statins led us to hypothesize that LSS increases endothelial responsiveness to statins. We demonstrate for the first time that treatment of mice with atorvastatin induces HO-1 expression in the aortic endothelium and that this occurs preferentially at sites exposed to LSS. In vitro, pre-conditioning human EC with an atheroprotective, but not an atheroprone waveform, significantly reduces the concentration of atorvastatin required to enhance HO-1-mediated cytoprotection against oxidant-induced injury. A synergistic relationship between LSS and statins is revealed, resulting in maximal Akt phosphorylation and dependence upon eNOS, Kruppel-like factor 2 (KLF2), and NF-E2-related factor-2 (Nrf2) activation. Actinomycin D, hydrogen peroxide (H2O2), paraformaldeyde, Triton X-100, trypan blue, and anti-α-tubulin antibody were from Sigma. Atorvastatin and simvastatin were from Merck Biosciences (Nottingham, UK) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) from Promega (Southampton, UK). NG-Nitro-l-arginine methyl ester (l-NAME) from BIOMOL (Plymouth Meeting, PA), leptin from R&D Systems, anti-phospho-Akt (Ser473) antibody from Cell Signaling (Beverly, MA), and anti-HO-1 antibodies were from Cambridge Bioscience (Cambridge, UK) and Stressgen (Victoria, BC). The nuclear extraction kit NE-PER Nuclear and Cytoplasmic Extraction Reagents were from Thermo Fisher Scientific Inc. Nrf2 activation in EC nuclear extracts was analyzed using an Nrf2 TransAMTM assay kit (Active Motif, Carlsbad, CA). Human umbilical vein EC (HUVEC) and human aortic EC (purchased from Promocell, Heidelberg, Germany) were cultured as described (20.Mason J.C. Ahmed Z. Mankoff R. Lidington E.A. Ahmad S. Bhatia V. Kinderlerer A. Randi A.M. Haskard D.O. Circ. Res. 2002; 91: 696-703Crossref PubMed Scopus (89) Google Scholar). The use of human EC was approved by Hammersmith Hospitals Research Ethics Committee (number 06/Q0406/21). Confluent EC monolayers (passage 3) on fibronectin-coated glass slides were exposed to control static conditions, high shear unidirectional laminar flow (12 dynes/cm2), or oscillatory flow (OF) with directional changes of flow at 1 Hz (± 5 dynes/cm2), for up to 48 h using a parallel-plate flow chamber (Cytodyne, La Jolla, CA) as described previously (21.Kinderlerer A.R. Ali F. Johns M. Lidington E.A. Leung V. Boyle J.J. Hamdulay S.S. Evans P.C. Haskard D.O. Mason J.C. J. Biol. Chem. 2008; 283: 14636-14644Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). To investigate synergy between LSS and statins, EC were exposed to static conditions or unidirectional LSS (12 dynes/cm2) for a total of 24 h. After 12 h, statin or vehicle control was added to the culture medium of static cells or to the medium in the flow apparatus via the injection port, whereas EC remained under conditions of continuous LSS. Cell viability was assessed by examination of EC monolayers using phase-contrast microscopy, cell counting, and estimation of trypan blue exclusion. Previously validated siRNA sequences targeting KLF2, HO-1, or scrambled control siRNA were transfected into HUVEC using oligofectamine-based transfection in endothelial basal medium 2 as described (18.Ali F. Hamdulay S.S. Kinderlerer A.R. Boyle J.J. Lidington E.A. Yamaguchi T. Soares M.P. Haskard D.O. Randi A.M. Mason J.C. J. Thromb. Haemost. 2007; 5: 2537-2546Crossref PubMed Scopus (79) Google Scholar): HO-1, sense, 5′UGCUGAGUUCAUGAGGAACUU-3′ and antisense, 5′-GUUCCUCAUGAACUCAGCAUU-3′; sense, 5′-CAUUGCCAGUGCCACCAAGUU-3′ and antisense, 5′-CUUGGUGGCACUGGCAAUGUU-3′; KLF2, sense, 5′-GCCCUACCACUGCAACUGGUU-3′ and antisense, 5′-CCAGUUGCAGUGGUAGGGCUU-3′; sense, 5′- GUUUGCGCGCUCAGACGAGUU-3′ and antisense, 5′- CUCGUCUGAGCGCGCAAACUU-3′. EC were cultured for 24 h in endothelial basal medium 2 and analyzed for target gene expression by quantitative RT-PCR or immunoblotting, which demonstrated up to 80% reduction in expression as reported (18.Ali F. Hamdulay S.S. Kinderlerer A.R. Boyle J.J. Lidington E.A. Yamaguchi T. Soares M.P. Haskard D.O. Randi A.M. Mason J.C. J. Thromb. Haemost. 2007; 5: 2537-2546Crossref PubMed Scopus (79) Google Scholar). The specificity of siRNA targeting was confirmed using a second set of sequences. Efficacy of siRNA was verified in each experiment. The recombinant adenovirus expressing dominant-negative (DN) Akt was a gift from Dr. C. Wheeler-Jones (Royal Veterinary College, London). The adenovirus expressing DN-Nrf2, which lacks the transactivation domain (Ad-Nrf2-DN) was provided by Dr. Jeffrey A. Johnson, University of Wisconsin, Madison, WI (22.Kraft A.D. Johnson D.A. Johnson J.A. J. Neurosci. 2004; 24: 1101-1112Crossref PubMed Scopus (456) Google Scholar). Adenoviruses were amplified in HEK-293A cells, purified, and titered using BD Adeno-X Purification and Rapid Titer Kits (BD Biosciences). HUVEC were infected by incubation with adenovirus in serum-free M199 for 2 h at 37 °C. The media was then changed to M199, 10% fetal bovine serum and HUVEC incubated overnight prior to experimentation. Infection of HUVEC with a β-galactosidase control adenovirus demonstrated a transfection efficiency of ≥95%. The optimal multiplicity of infection for the DN-Nrf2 and DN-Akt adenoviruses was determined by immunoblotting (not shown). The plasmid pHO-1-Luc was a gift from J. Alam (Alton Ochsner Medical Foundation, New Orleans, LA). EC were transfected in triplicate with pGL3-basic or pHO-1-Luc using microporation technology (Digital Bio, Seoul, Korea) as described previously (18.Ali F. Hamdulay S.S. Kinderlerer A.R. Boyle J.J. Lidington E.A. Yamaguchi T. Soares M.P. Haskard D.O. Randi A.M. Mason J.C. J. Thromb. Haemost. 2007; 5: 2537-2546Crossref PubMed Scopus (79) Google Scholar). HUVEC were loaded with 5 μm 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) (Invitrogen) for 30 min at 37 °C. ECs were incubated in serum-free M199 with H2O2 (5 μm) for 30 min or 100 ng/ml leptin for 2 h and then washed with ice-cold phosphate-buffered saline. Intracellular dihydrodichlorofluorescein (H2DCF) was oxidized to 2,7-dichlorofluorescein and quantified by flow cytometry. H2O2 (50 μm) was used to induce cellular injury and this was quantified using either: (i) trypan blue exclusion or (ii) a MTT assay. Cell numbers were not significantly altered by any of the treatment conditions prior to addition of H2O2. Quantitative real-time PCR was performed using an iCycler (Bio-Rad). β-Actin, glyceraldehyde-3-phosphate dehydrogenase, and hypoxanthine-guanine phosphoribosyltransferase were used as housekeeping genes, with data calculated in relation to the β-Actin gene and verified with glyceraldehyde-3-phosphate dehydrogenase and hypoxanthine-guanine phosphoribosyltransferase. DNase-I-digested total RNA (1 μg) was reverse transcribed using 1 μm oligo(dT) and Superscript reverse transcriptase (Invitrogen). cDNA was amplified in a 25-μl reaction containing 5 μl of cDNA template, 12.5 μl of iSYBR supermix, 0.5 pm sense and antisense gene-specific primers, and double distilled H2O. Primer sequences used were: KLF2 forward, 5′-CTTTCGCCAGCCCGTGCCGCG-3′, KLF2 reverse, 5′-AAGTCCAGCACGCTGTTGAGG-3′; HO-1 forward, 5′-CTTCTTCACCTTCCCCAACA-3′, HO-1 reverse, 5′-TTCTATCACCCTCTGCCTGA-3′; Nrf2 forward, 5′-AAACCAGTGGATCTGCCAAC-3′, Nrf2 reverse, 5′-GACCGGGAATATCAGGAACA-3′ (23.Leonard M.O. Kieran N.E. Howell K. Burne M.J. Varadarajan R. Dhakshinamoorthy S. Porter A.G. O'Farrelly C. Rabb H. Taylor C.T. FASEB J. 2006; 20: 2624-2626Crossref PubMed Scopus (101) Google Scholar); TM forward, 5′-TTGTGGAATTGGGAGCTTGG-3′, TM reverse, 5′-TCTCATGAACTGGATGGGGTG-3′ (24.van Thienen J.V. Fledderus J.O. Dekker R.J. Rohlena J. van Ijzendoorn G.A. Kootstra N.A. Pannekoek H. Horrevoets A.J. Cardiovasc. Res. 2006; 72: 231-240Crossref PubMed Scopus (102) Google Scholar); and eNOS forward, 5′- TGGCTTTCCCTTCCAGTTC-3′, eNOS reverse 5′-AGAGGCGTTTTGCTCCTTC-3′ (24.van Thienen J.V. Fledderus J.O. Dekker R.J. Rohlena J. van Ijzendoorn G.A. Kootstra N.A. Pannekoek H. Horrevoets A.J. Cardiovasc. Res. 2006; 72: 231-240Crossref PubMed Scopus (102) Google Scholar). Cycling parameters were 3 min at 95 °C, and 40 cycles of 95 °C for 10 s and 56 °C for 45 s. Immunoblotting was performed as described (25.Mason J.C. Steinberg R. Lidington E.A. Kinderlerer A.R. Ohba M. Haskard D.O. J. Biol. Chem. 2004; 279: 41611-41618Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). HUVEC were incubated with atorvastatin for up to 72 h prior to lysis, SDS-PAGE, and transfer to polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA). Immunoblots were probed with primary antibodies overnight at 4 °C, followed by appropriate secondary reagents for 1 h at room temperature and developed with a chemiluminescence substrate (Amersham Biosciences). To ensure equivalent sample loading, protein content was determined using the Bio-Rad Dc protein assay (Bio-Rad) and membranes were stripped and re-probed with a control antibody. Integrated density values were obtained with an Alpha Innotech ChemiImager 5500 (Alpha Innotech, San Leandro, CA). The measurement of intracellular cholesterol was carried out using a procedure previously described in detail by Wang et al. (26.Wang Y. Karu K. Griffiths W.J. Biochimie. 2007; 89: 182-191Crossref PubMed Scopus (37) Google Scholar). C57BL/6 mice were from Harlan Olac (Bicester, Oxford, UK) and housed under controlled climactic conditions in microisolator cages with autoclaved bedding. Irradiated food and drinking water were readily available. All animals were housed and studied according to UK Home Office guidelines. Sentinel mice were housed alongside test animals and regularly screened for a standard panel of murine pathogens. En face confocal microscopy was used to assess changes in the expression of HO-1 in the murine aortic vascular endothelium. C57BL/6 mice (n = 6) were injected intraperitoneally with atorvastatin (5 mg/kg) or vehicle alone and sacrificed 24 h later by CO2 inhalation, followed by perfusion fixation with 2% formalin and harvesting of aortae. Fixed aortae were treated with an HO-1 specific primary antibody (Cambridge Biosciences) and an Alexa Fluor 568-conjugated secondary antibody. Stained vessels were mounted prior to visualization of endothelial surfaces en face using confocal laser scanning microscopy (LSM 510 META; Zeiss, Oberkochen, Germany). Changes in the expression of HO-1 in murine aortic EC located in regions of the lesser curvature exposed to disturbed flow and both the greater curvature and descending aorta exposed to laminar flow were quantified as described (27.Hajra L. Evans A.I. Chen M. Hyduk S.J. Collins T. Cybulsky M.I. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 9052-9057Crossref PubMed Scopus (465) Google Scholar). EC were identified by co-staining with anti-CD31 antibody conjugated to the fluorophore fluorescein isothiocyanate (Invitrogen). Nuclei were identified using a DNA-binding probe with far-red emission (Draq5; Biostatus, Leicester, UK). Isotype-matched monoclonal antibodies against irrelevant antigens were used as experimental controls for specific staining. HO-1 protein expression was quantified by image analysis of fluorescence intensity in 100 cells in at least 3 distinct sites using Image J software. EC fluorescence was measured above a threshold intensity defined by background fluorescence. Data were grouped according to treatment and analyzed using GraphPad Prism software (San Diego, CA) and the analysis of variance with Bonferroni correction or an unpaired Student's t test. Data are expressed as the mean of individual experiments ± S.E. Differences were considered significant at p values of <0.05. To establish whether statins increase endothelial HO-1 expression in vivo, C57Bl/6 mice were treated with atorvastatin for 24 h. Changes in HO-1 expression were quantified by en face confocal microscopy of the aortic endothelium, with endothelial cells identified by CD31 staining. As shown in Fig. 1A, treatment with atorvastatin induced a significant increase in HO-1 expression in murine aortic endothelium at a site with low probability of developing atherosclerotic lesions (27.Hajra L. Evans A.I. Chen M. Hyduk S.J. Collins T. Cybulsky M.I. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 9052-9057Crossref PubMed Scopus (465) Google Scholar). In contrast, although HO-1 induction was detectable, EC located in the lesser curvature of the aorta, which has a high probability of developing lesions, were relatively refractory to atorvastatin treatment (Fig. 1B). Quantification by image analysis confirmed that HO-1 induction at high probability sites was significantly less than that at low probability sites (Fig. 1C). Statins and unidirectional LSS separately induce EC HO-1 expression in vitro. An established physiologic hemodynamic environment was therefore used to explore the influence of LSS on statin responsiveness. As expected, treatment of HUVEC with either 2.5 μm atorvastatin under static conditions, or exposure of HUVEC to LSS for 24 h, significantly increased HO-1 mRNA levels (Fig. 2A). Pre-conditioning of EC with LSS (12 dynes/cm2) for 12 h prior to addition of atorvastatin and continuation of culture under LSS for a further 12 h resulted in an additive increase in HO-1 mRNA (Fig. 2A). Reduction of the atorvastatin concentration applied to static-cultured EC to 0.6 μm led to loss of HO-1 induction. In contrast, a significant increase of HO-1 mRNA was still seen when this concentration was applied to EC pre-conditioned by LSS, with 0.6 μm atorvastatin inducing a 14-fold increase in HO-1 mRNA (Fig. 2B). A dose-response study confirmed synergy between LSS (12 dynes/cm2) and atorvastatin (1.25 and 0.6 μm), and this was lost with 0.3 μm atorvastatin (Fig. 2C). Moreover, further studies using an HMOX1 promoter reporter construct confirmed synergy between atorvastatin (0.6 μm) and LSS, as indicated by relative luciferase activity (Fig. 2D). A comparable synergistic response was also seen with simvastatin (supplementary Fig. S1A), whereas immunoblotting confirmed enhanced induction of HO-1 protein by 0.6 μm atorvastatin in LSS-conditioned EC (Fig. 2E). The importance of the duration of LSS was revealed by loss of the synergistic response when pre-conditioning was reduced to 6 h (supplementary Fig. S1, B and C). Subsequent experiments performed with human aortic EC, to represent an endothelial surface affected by atherosclerosis, demonstrated that they responded similarly to HUVEC with synergy observed between LSS and atorvastatin (0.6 μm), resulting in a 13-fold increase in HO-1 mRNA (supplementary Fig. S1D). To compare the effect of atheroprotective and atheroprone waveforms on responsiveness to statins, EC were exposed to LSS (12 dynes/cm2) or OF (±5 dynes/cm2 at 1 Hz) (21.Kinderlerer A.R. Ali F. Johns M. Lidington E.A. Leung V. Boyle J.J. Hamdulay S.S. Evans P.C. Haskard D.O. Mason J.C. J. Biol. Chem. 2008; 283: 14636-14644Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). A 10-fold increase in HO-1 expression was seen in EC exposed to 24 h LSS, whereas HO-1 mRNA induction was reduced to 3-fold in EC exposed to OF. Furthermore, OF-conditioned EC failed to demonstrate a synergistic relationship with atorvastatin (Fig. 2F). To investigate the functional relevance of HO-1 induction, EC were exposed to free radical-induced injury. Atorvastatin (0.6 μm) failed to protect static-cultured HUVEC exposed to H2O2 (50 μm). However, EC exposed to LSS for 24 h were protected by 50%, and this was significantly enhanced by atorvastatin (0.6 μm) (Fig. 3A). An oxygen radical-sensitive fluorescent probe (CM-H2DCFDA) was used to explore the ability of atorvastatin to modulate oxidative stress. LSS alone led to low-level oxidant generation, whereas exposure of EC to H2O2 (5 μm) induced a maximal response (Fig. 3B). Pre-treatment of static-cultured EC with atorvastatin (0.6 μm) failed to protect, whereas LSS alone significantly reduced H2O2 generated oxidative stress. However, as predicted, maximal protection was seen in LSS-conditioned EC treated with atorvastatin (Fig. 3B). Treatment with leptin also increased EC oxidant generation by 5-fold. LSS was again protective, with maximal reduction in leptin-induced oxidative stress seen in those cells exposed to both LSS and atorvastatin (Fig. 3C). To determine the role of HO-1 in the cytoprotective response, HUVEC were transfected with HO-1-specific or control siRNA. HO-1 siRNA reduced mRNA levels by 80% (supplementalFig. 2A) (18.Ali F. Hamdulay S.S. Kinderlerer A.R. Boyle J.J. Lidington E.A. Yamaguchi T. Soares M.P. Haskard D.O. Randi A.M. Mason J.C. J. Thromb. Haemost. 2007; 5: 2537-2546Crossref PubMed Scopus (79) Google Scholar). Interference with HO-1 expression significantly reduced cytoprotection against H2O2-induced cell death afforded by atorvastatin in LSS-conditioned EC, from 75 to 40% (Fig. 3D), suggesting HO-1 is an important but not necessarily unique protective mechanism. In line with reduced HO-1 induction, exposure of EC to an atheroprone OF pattern revealed markedly attenuated protection against H2O2. Moreover, atorvastatin failed to enhance cytoprotection against oxidant injury in this setting (Fig. 3E). As shown in Fig. 4A, exposure of HUVEC to LSS, or treatment of static-cultured EC with atorvastatin (0.6 μm), resulted in a modest reduction in intracellular cholesterol, which did not reach significance. However, consistent with the effect on HO-1 expression, pre-conditioning of EC with LSS prior to addition of atorvastatin resulted in a significant fall in intracellular cholesterol up to 60%. These data, combined with that in Fig. 2, suggest that EC exposure to LSS significantly enhances responsiveness to statins. To determine whether LSS and atorvastatin regulate HO-1 expression post-transcriptionally, EC were exposed to LSS in the presence or absence of atorvastatin, prior to addition of actinomycin D (2 μg/ml) and analysis of HO-1 by quantitative RT-PCR. Treatment with actinomycin D resulted in less than 5% cell death as estimated by trypan blue exclusion studies. The rapid decay of HO-1 mRNA in static-cultured EC was not delayed by atorvastatin. In contrast, LSS increased HO-1 mRNA stability and this delay in degradation was
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