Angiotensin II enhances AT1-Nox1 binding and stimulates arterial ...

Am J Physiol Heart Circ Physiol 303: H282–H296, 2012. First published May 25, 2012; doi:10.1152/ajpheart.00231.2012.

Angiotensin II enhances AT1-Nox1 binding and stimulates arterial smooth muscle cell migration and proliferation through AT1, Nox1, and interleukin-18 Anthony J. Valente,1 Tadashi Yoshida,2 Subramanyam N. Murthy,3 Siva S. V. P. Sakamuri,2 Masato Katsuyama,4 Robert A. Clark,1 Patrice Delafontaine,2 and Bysani Chandrasekar2,5 1

Medicine, University of Texas Health Science Center and South Texas Veterans Health Care System, San Antonio, Texas; Heart and Vascular Institute, Tulane University School of Medicine, New Orleans, Louisiana; 3Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana; 4Radioisotope Center, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto, Japan; and 5Research Service, Southeast Louisiana Veterans Health Care System, New Orleans, Louisiana 2

Submitted 21 March 2012; accepted in final form 15 May 2012

Valente AJ, Yoshida T, Murthy SN, Sakamuri SS, Katsuyama M, Clark RA, Delafontaine P, Chandrasekar B. Angiotensin II enhances AT1-Nox1 binding and stimulates arterial smooth muscle cell migration and proliferation through AT1, Nox1, and interleukin18. Am J Physiol Heart Circ Physiol 303: H282–H296, 2012. First published May 25, 2012; doi:10.1152/ajpheart.00231.2012.—The redox-sensitive transcription factors NF-␬B and activator protein-1 (AP-1) are critical mediators of ANG II signaling. The promitogenic and promigratory factor interleukin (IL)-18 is an NF-␬B- and AP-1responsive gene. Therefore, we investigated whether ANG II-mediated smooth muscle cell (SMC) migration and proliferation involve IL-18. ANG II induced rat carotid artery SMC migration and proliferation and IL-18 and metalloproteinase (MMP)-9 expression via ANG II type 1 (AT1) receptor. ANG II-induced superoxide generation, NF-␬B and AP-1 activation, and IL-18 and MMP-9 induction were all markedly attenuated by losartan, diphenyleneiodonium chloride (DPI), and Nox1 knockdown. Similar to ANG II, addition of IL-18 also induced superoxide generation, activated NF-␬B and AP-1, and stimulated SMC migration and proliferation, in part via Nox1, and both ANG II and IL-18 induced NOX1 transcription in an AP-1dependent manner. AT1 physically associates with Nox1 in SMC, and ANG II enhanced this binding. Interestingly, exogenous IL-18 neither induced AT1 binding to Nox1 nor enhanced the ANG II-induced increase in AT1/Nox1 binding. Importantly, IL-18 knockdown, or pretreatment with IL-18 neutralizing antibodies, or IL-18 binding protein, all attenuated the migratory and mitogenic effects of ANG II. Continuous infusion of ANG II for 7 days induced carotid artery hyperplasia in rats via AT1 and was associated with increased AT1/ Nox1 binding (despite lower AT1 levels); increased DPI-inhibitable superoxide production; increased phospho-IKK␤, JNK, p65, and cJun; and induction of IL-18 and MMP-9 in endothelium-denuded carotid arteries. These results indicate that IL-18 amplifies the ANG II-induced, redox-dependent inflammatory cascades by activating similar promitogenic and promigratory signal transduction pathways. The ANG II/Nox1/IL-18 pathway may be critical in hyperplastic vascular diseases, including atherosclerosis and restenosis. renin-angiotensin-aldosterone system; mitogenesis; migration; atherosclerosis; restenosis; angiotensin II type 1 receptor

for cell growth, migration, and proliferation. Chronically elevated levels of angiotensin (ANG) II play a role in atherosclerosis and restenosis (15, 30). ANG II induces oxidative stress, proinflammatory cytokine, and chemokine expression, endothelial cell death, smooth muscle cell HYPERTENSION IS A POTENT STIMULUS

Address for reprint requests and other correspondence: B. Chandrasekar, Heart and Vascular Institute, Tulane Univ. School of Medicine, 1430 Tulane Ave., SL-48, New Orleans, LA 70112. H282

(SMC) growth, migration, and proliferation, and infiltration of inflammatory cells into the vessel wall (8, 11, 24, 41). The pleiotropic cytokine interleukin (IL)-18 also plays a causal role in atherosclerosis. In animal models, increased expression of IL-18 is associated with the development and progression of atherosclerosis. Its neutralization inhibits neointimal formation in a rat model of vascular injury (25). Systemic levels of IL-18 are increased in subjects with coronary artery disease and correlate positively with intima-media thickness (51). IL-18 expression is elevated in atherosclerotic lesions, particularly in unstable plaques (28). By inducing other proinflammatory cytokines, chemokines, and adhesion molecules, it may further amplify the inflammatory cascade. IL-18 also induces metalloproteinase (MMP) expression (4, 39). MMPs degrade extracellular matrix and promote cellular migration and subsequent proliferation. We previously reported that IL-18 induces MMP9 transcription, mRNA expression, and enzymatic activity in human coronary artery SMC (4). Similar to ANG II, IL-18 can also induce endothelial cell death (5) and SMC migration and proliferation (4, 40), suggesting that IL-18 may mediate the hypertrophic and hyperplastic effects of ANG II. Although ANG II signals via the seven transmembrane G-protein-coupled ANG II type 1 and 2 (AT1 and AT2) receptors, it is thought that the majority of its proatherogenic, proinflammatory, and pro-oxidant effects are mediated via AT1 (9, 35, 36). ANG II is a potent inducer of oxidative stress, and several studies (10, 33, 49) have established a central role for the Nox members of the NADPH oxidase family in SMC migration and proliferation. Among the seven family members of the NADPH oxidases (Nox1 to 5, Duox1 and 2), human vascular SMC typically express Nox1 and Nox4 isoforms with differential localization in the cellular compartments. For example, while Nox1 is located in the plasma membrane and is inducible, Nox4 is constitutively active and found in several subcellular compartments, including nucleus, endoplasmic reticulum, and mitochondria (10). Interestingly, while both generate reactive oxygen species (ROS), the species of ROS may differ. Nox1 activity results in superoxide generation, whereas hydrogen peroxide is usually the only detectable product of Nox4 activity (10). In a mouse model, Schröder et al. (43) recently reported that Nox4 is predominantly expressed in murine aortic and carotid artery endothelial cells. They demonstrated that while normal vessels express Nox4 mRNA and protein, its expression levels were markedly decreased following enzymatic removal of endothelial cells from those arteries. http://www.ajpheart.org

AT1/NOX1 AND IL-18 MEDIATE ANG-II-INDUCED SMC MIGRATION

They concluded that Nox4 expression is higher in endothelium than in smooth muscle cells in normal murine arteries. Further, Clempus et al. (6) demonstrated that while Nox1 is associated with, and promotes, SMC proliferation, Nox4 plays a role in the maintenance of a differentiated phenotype. Importantly, while ANG II induces Nox1 expression and activity in SMC, it inhibits Nox4 expression (10). Therefore, we hypothesize that ANG II-mediated IL-18 induction and SMC migration and proliferation, are mediated through Nox1-dependent ROS generation. As second messengers, ROS activate several cellular signal transduction pathways, including two critical oxidative stressresponsive transcription factors NF-␬B and activator protein-1 (AP-1). We previously demonstrated that Il18 is an NF-␬B and AP-1 responsive gene (2). In addition, IL-18 is a potent activator of NF-␬B and AP-1 in SMC (4). Thus upon induction IL-18 may regulate its own expression and that of other NF-␬B- and AP-1-responsive inflammatory cytokines, chemokines, adhesion molecules, and MMPs and further amplify the inflammatory cascade. Therefore, we investigated the effects of ANG II on NF-␬B and AP-1 activation in primary SMC isolated from rat carotid artery and defined the upstream signaling pathways involved in their activation. Here we show that ANG II is a potent inducer of hyperplasia both in vitro and in vivo. In vitro, it induced SMC migration and proliferation via AT1/Nox1-dependent ROS generation. ANG II induced NF-␬B and AP-1 activation and IL-18 and MMP-9 expression. Similar to ANG II, IL-18 enhanced ROS generation, NF-␬B and AP-1 activation, and SMC migration and proliferation via Nox1. Notably, we demonstrate for the first time that AT1 physically associates with Nox1 in vitro and in vivo and that ANG II, but not IL-18, enhances their binding in vivo. These results were recapitulated in carotid arteries from ANG II-infused rats. Our results indicate that ANG II stimulates SMC migration and proliferation via AT1/Nox1dependent IL-18 induction, and this process may involve the physical association of AT1/Nox1. The AT1/Nox1/IL-18 pathway may be critical in hyperplastic vascular diseases, including atherosclerosis and restenosis. MATERIALS AND METHODS

Human ANG II (no. A9525), AT1 antagonist losartan potassium (no. 61188; 10 mM for 1 h), AT2 antagonist (PD 123319; 10 mM for 1 h), diphenyleneiodonium chloride (D-2926), doxorubicin (1 ␮M in water), Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid; 5 mM for 1 h), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Tempol (1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine, no. 3082; 1 mM in DMSO), a membrane-permeable superoxide scavenger, was purchased from TOCRIS Bioscience (Ellisville, MO). MMP2/9 inhibitor (MMP2/9I; no. 444241), Marimastat (no. 444289), GM 6001 (no. 364205), and DMSO were purchased from EMD Biosciences (Philadelphia, PA). The following antibodies were used: ␣-sarcomeric actin, Nox1 (SAB4200097), and Myc (Sigma-Aldrich); phospho-p65 (Ser536; no. 3033), phospho-IKK␤ (Ser176/Ser180; no. 2697), IKK␤ (no. 2370), MMP-9 (3852S), phospho-c-Jun (no. 9261), JNK (no. 9252), phospho-JNK (Thr183/Tyr185; no. 9251), ␣-tubulin (no. 2144), GAPDH (no. 2118), and lamin A/C (no. 2032; Cell Signaling Technology, Beverly, MA); IL-18 (no. AF521), control IgG (no. AB-108-C), IL-18BP-Fc (no. 119-BP-100), and Fc (R&D Systems, Minneapolis, MN); and rabbit anti-human AT1 polyclonal antibodies (no. sc-1173; Santa Cruz Biotechnology, Santa Cruz, CA). Recombinant rat PDGF-BB (no. 520-BB-050) was from R&D Sys-

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tems. Rat IL-18 ELISA kit (no. EK0592) was from InsightGenomics (Falls Church, VA). Enhanced chemiluminescence detection kit was from Amersham Pharmacia Biotech. The Diogenes reagent was from National Diagnostics (Cherry Hill, NJ). Cell Death Detection ELISAPLUS (no. 11774425001) was from Roche Applied Science (Indianapolis, IN). All tissue culture supplies were from Invitrogen (Carlsbad, CA). Stress-activated protein kinase/JNK assay kit (no. 9810) was from Cell Signaling Technology. BD BioCoat Matrigel invasion chambers (no. 354481) were from BD Biosciences Discovery Labware (Billerica, MA). ALZET miniosmotic pumps were purchased from DURECT (Cupertino, CA). At the indicated concentrations and for the duration of treatment, the pharmacological inhibitors failed to modulate SMC morphology, viability, or adherence to culture dishes (data not shown). Animals This investigation conforms to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (DRR/National Institutes of Health, 1996), and all protocols were approved by the Institutional Animal Care and Use Committees of the University of Texas Health Science Center (San Antonio, TX) and Tulane University (New Orleans, LA). For the in vivo studies, normotensive male Sprague-Dawley rats (⬃3 m of age, ⬃200 g; Charles River Laboratories International, Wilmington, MA) were used. Animals were allowed 7 days to acclimatize and then trained for systolic blood pressure (SBP) measurement using a tail-cuff method without anesthesia (CODA noninvasive blood pressure system; Kent Scientific, Torrington, CT; ref. 44). The animals were divided into four groups: group 1: saline alone. Saline was infused for 7 days via subcutaneously implanted (midscapular region) ALZET miniosmotic pumps (n ⫽ 12). Group 2: saline ⫹ losartan; 24 h before saline infusion, a subgroup of animals was administered with losartan, a selective AT1 blocker, via drinking water (400 mg/l; 40 mg·kg body wt⫺1·day⫺1; n ⫽ 6). Group 3: ANG II alone; ANG II was infused at 700 ␮g·kg⫺1·day⫺1 for 7 days via miniosmotic pumps (n ⫽ 6). Group 4: ANG II ⫹ losartan; 24 h before ANG II infusion, a subgroup of animals was administered with losartan potassium via drinking water (n ⫽ 6). Before death, blood pressure and body weights were recorded in all four groups (44). Heart and left ventricles were weighed to quantify hypertrophy. The right and left carotid arteries were rapidly excised and rinsed in ice-cold physiological saline, and a small (2 mm) representative area of the right carotid artery was fixed in 10% buffered formaldehyde, embedded in paraffin, sectioned, and used for histomorphometric analysis. Computerized digital microscopic software (Image-Pro Plus 4) was used to obtain measurements of the intimal and medial areas. The endothelium was denuded from the rest of the vessel and frozen for biochemical and molecular analysis. Our pilot experiments demonstrated that ANG II infusion increases medial thickness in both right and left carotid arteries to a similar extent (data not shown). Cell Culture SMC were isolated from the right and left carotid arteries from naïve rats by an explant method described previously (40). In brief, vessels were opened longitudinally, scraped free of endothelium, and cut into small pieces. The endothelium-denuded tissue was placed with luminal surface facing down on a 0.1% gelatin-coated culture flask containing DMEM/F-12 ⫹ 20% FCS ⫹ antibiotics (100 U/ml penicillin, 100 ␮g/ml streptomycin, and 0.25 ␮g/ml amphotericin B) and cultured at 37°C in a humidified atmosphere of 95% air-5% CO2. After 12–14 days, the SMC outgrowths from the explants were trypsinized and replated in tissue culture dishes. Cells were pooled from both vessels and used between passages 4 –7. Around 96% of cells were positive for smooth muscle cell ␣-actin. At ⬃70% confluency, the cells were washed twice with serum-free medium and then maintained in DMEM/F12 with 0.1% BSA for 48 h before use.

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00231.2012 • www.ajpheart.org

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Measurement of Superoxide ·⫺

In whole cells. Superoxide (O2 ) generation was quantified using the lucigenin-enhanced chemiluminescence assay as previously described (22, 44). In brief, SMC were rinsed twice in PBS and resuspended in Krebs/HEPES buffer (in mM: 115 NaCl, 20 HEPES, 1.17 K2HPO4, 1.17 MgSO4, 4.3 KCl, 1.3 CaCl2, 25 NaHCO3, and 11.7 glucose pH 7.4) and contained no NADPH in the medium. After incubation at 37°C for 30 min, the tubes were placed in a Sirius luminometer (Berthold Detection Systems, Pforzheim, Germany). Dark-adapted lucigenin (5 ␮M) and either ANG II (10⫺7 M) alone or ANG II combined with diphenyleneiodonium chloride (DPI), Tiron, or Tempol were added. Luminescence was measured for 10 s with a delay of 5 s. After background luminescence was subtracted, results were expressed as relative light units (RLU) per second per 5 ⫻ 104 cells. Studies were also performed using SMC infected with Ad.CuZnSOD [multiplicity of infection (MOI) 100 for 24 h] or transfected with small interfering (si)RNA for Nox1 knockdown (50 nM for 72 h) before assay. Superoxide generation was also measured using a luminol-based chemiluminescence assay (Diogenes, National Diagnostics) as previously described (13). Following trypsinization, cells were washed once in PBS, resuspended at 1⫻104/ml in PBS containing 10 mM glucose (PBS-G) and kept on ice until assayed. For the assay, 100 ␮l of the luminol reagent were mixed with 1⫻104 cells and incubated at 37°C for 2– 4 min. Superoxide generation was stimulated by the addition of ANG II in PBS-G, with and without inhibitors as described above. Chemiluminescence was measured every 30 – 60 s using a Turner Designs 20/20 luminometer and a 5-s integration time. In SMC homogenates. We also measured superoxide generation in SMC homogenates (100 ␮g/sample) using the lucigenin-enhanced chemiluminescence assay as detailed above, but the reaction mixture contained NADPH (100 ␮M; Ref. 22). Studies were also performed using homogenates prepared from SMC treated with DPI or transfected with Nox1 siRNA (50 nM for 72 h) before ANG II addition. In carotid artery homogenates. Superoxide generation in carotid artery homogenates was measured by lucigenin-enhanced chemiluminescence (22) as detailed in SMC homogenates. Adenoviral Transduction Adenovirus encoding green fluorescent protein (Ad.GFP), kinasedeficient (kd) I␬␬␤ (Ad.kdIKK␤), dominant negative (dn) JNK (Ad.dnJNK1), dnc-Jun (Ad.dnc-Jun), and dnRac1 (Ad.N17rac1) were all described before (2–5). Adenoviral CuZnSOD (Ad.CuZnSOD) was obtained from Gene Transfer Vector Core at the University of Iowa after permission from John F. Engelhardt (University of Iowa College of Medicine, Iowa City, IA). SMC were infected with adenoviruses at indicated MOI at ambient temperature. After 1 h, the adenovirus-containing medium was replaced with medium containing 0.5% BSA. Twenty hours later, SMC were incubated with ANG II. At the indicated MOI, infection with adenovirus did not affect SMC viability, adherence, shape, or growth (data not shown). RNA Interference SMC were transfected with Nox1 siRNA [NM_053683.1; sense: r(GAGUGGGAUAAGUAUGAAA)dTdT and antisense: r(UUUCAUACUUAUCCCACUC)dTdT; Rn_Nox1_1Flexitube siRNA, SI019 208435, Qiagen, Valencia, CA]. A nonsilencing siRNA oligonucleotide sequence (NS siRNA; All Stars Negative Control siRNA, no. 1027280, Qiagen) consisting of a scrambled sequence that does not recognize any mammalian gene was used as a negative control. Nontargeting siRNA and siRNA that specifically targets MMP-9 (Mission siRNA; SASI_Rn01_00037990) and IL-18 (SASI_Rn02_00261951) were purchased from Sigma-Aldrich. SMC were transfected with the indicated siRNA (Nox1, 50 nM; IL-18, 50 nM, MMP-9, 5 nM) using the Neon transfection system (MPK-5000; Invitrogen) with the following param-

eters: pulse voltage 1,475 V, pulse width 20 ms, pulse number 2, and the tip type 10 ␮l. Cell viability was 97%. Seventy-two hours after transfection, knockdown of Nox1, IL-18, and MMP-9 was confirmed by immunoblotting. No off-target effects were observed. Promoter-Reporter Assays The 0.146 kb NOX1 5’-flanking region upstream of the transcription initiation site in pGL3-Basic vector and the construct with mutation of the AP-1 binding site were previously described (29). The inducible murine Il18 gene contains two TATA-less functional regions upstream of exon 1 (⫺2,505 to ⫹61 nt) and NF-␬B and AP-1 response elements (2). The 0.726-kb human MMP9 promoter-reporter vector has also been described previously (4). pGL3-Basic served as a vector control. SMC were transiently transfected with the reporter vector (3 ␮g) using the Neon transfection system. Forty-eight hours later, cells were treated with ANG II (10⫺7M for 12 h). The Renillaluciferase reporter plasmid (100 ng; pRL-TK vector; Promega, Madison, WI) was used as an internal control. The transfection efficiency of SMC, determined using pEGFP-N1 vector (Clontech, BD Biosciences, Palo Alto, CA), was 69%. Cell viability after transfection was 93%, as determined using trypan blue dye exclusion. Luciferase activity was determined using the Promega Biotech dual-luciferase reporter assay system. Luciferase reporter data were normalized for transfection efficiency using the corresponding Renilla-luciferase values and expressed as mean relative stimulation ⫾ SE for a representative experiment from three separate and independent experiments with each performed in triplicate. Transcription Factor Activation by EMSA NF-␬B and AP-1 DNA binding activities in nuclear protein extracts were analyzed by EMSA using MMP9 gene-specific consensus (NF-␬B, sense, 5=-CTGCGGAAGACAGGggGTTGCCCCAGTGGAATTCCC-3=; AP-1, sense, 5=-CTGACCCCTGAGTcaGCACTT-3=) and mutant (NF-␬B, sense, 5=-CTGCGGAAGACAGGccGTTGCCCCAGTGGAATTCCC-3=; AP-1, sense, 5=-CTGACCCCTGAGTtgGCACTT-3=) primers (4). Specificity of TF binding activity was verified in competition experiments as follows: mutant: competition with respective mutant TF (NF-␬B or AP-1) oligonucleotide; nuclear protein extracts from IL-18-treated (1 ng/ml for 1 h) SMC were preincubated with 50-fold molar excess of unlabeled double-stranded mutant oligonucleotide followed by the addition of TF-specific probe; consensus: nuclear protein extracts from IL-18 treated (1 ng/ml) SMC were preincubated with 50-fold molar excess unlabeled consensus double stranded oligonucleotide encompassing the TF binding motif followed by the addition of TF-specific probe; and no protein: no protein extract but contained [␥-32P]ATP-labeled TF-specific probe. mRNA Expression by Real-Time Quantitative PCR and Northern blot analysis IL-18 and MMP-9 mRNA expression was analyzed by RT-quantitative (q)PCR. IL-18 mRNA expression was also analyzed by Northern blotting. Binding Interaction of AT1 with Nox1 In Vitro by GST Pull-Down Assays Rat Nox1 cDNA was kindly provided by Kathy K. Griendling (Emory University, Atlanta, GA). The full-length cDNA was reamplified and cloned into pCR2.1-TOPO, excised with NheI and XbaI, and cloned into the vector pcDNA3.1/Zeo(⫺). The interaction in vitro between the whole Nox1 molecule and the C-terminal cytoplasmic domain of rat AT1 [GST-AT1 (303–359)] was carried out as described previously (44). In addition, interaction studies were carried out between the C-terminal cytoplasmic domain of Nox1 fused to maltose-binding protein [MBP-Nox1(289 –563)] and GST-AT1(303–359)..



Binding interactions were carried out between GST and GSTATI(303–359) fusion protein immobilized on GSH-Sepharose (GE Health Care) and in vitro synthesized [35S]-labeled rat Nox1, MBPNox1(289 –563), and MBP. Volumes of GST fusion protein lysates, previously determined to give equal loading of the GSH-Sepharose, were mixed for 30 min at room temperature with 50 ␮l of a 50% suspension of washed GSH-Sepharose in PBS (145 mM NaCl and 10 mM Na2HPO4 pH 7.3). The suspensions were washed three times each with PBS and PBS containing 0.05% Triton X-100 and resuspended to 480 ␮l in PBS/0.05% Triton X-100. Rat Nox1, MBP-Nox1(289 –563), and MBP were synthesized and labeled in vitro with L-[35S]methionine (PerkinElmer NEG709A, 1,175 Ci/mmol) using the TNT coupled reticulocyte lysate system (Promega) and T7 RNA polymerase. A 20-␮l aliquot of the labeling reaction was mixed with 480 ␮l of the resuspended immobilized GST fusion protein for 30 min at room temperature. The gel pellet was washed three times with PBS/0.05% TX-100 and treated with 50 ␮l 2X SDS treatment buffer on ice for 20 min. After centrifugation for 5 min at 14,000 rpm, an aliquot of the supernatant (10 ␮l) was separated on 9% SDS-PAGE and analyzed by fluorography. For comparison, an aliquot of the labeled proteins equivalent to 5% of the input was included on the gel. To confirm equal loading of the GST proteins, 1 ␮l of the supernatant was separated on 9% SDS-PAGE and the gel was stained for protein (Imperial Protein Stain, ThermoScientific). Binding Interaction of AT1 with Nox1 by Immunoprecipitation and Immunoblotting For immunoprecipitation, equal amounts of membrane extracts or whole cell lysates were incubated overnight with specific antibodies attached to agarose beads at 4°C under slow rotation. After being washed three times in a buffer containing 50 mM Tris·HCl, 150 mM NaCl, and 0.1% Nonidet P-40, the bound proteins were eluted from the beads by boiling in SDS sample buffer for subsequent SDS-PAGE and immunoblotting. Immunoprecipitation/immunoblot studies were also performed using postadsorption supernatants. The AT1 and Nox1 antibodies used in immunoprecipitation and immunoblotting are detailed above. Immunoblotting The methods of preparation of whole cell homogenates, cytoplasmic, and nuclear protein extraction, immunoblotting, detection of the immunoreactive bands by enhanced chemiluminescence (ECL Plus; GE Healthcare), and their quantification by densitometry were all described before (2–5). ␣-Tubulin (whole cell homogenates), GAPDH (cytoplasmic), and lamin A/C (nuclear) served as loading and purity controls. Cell Migration Cultured SMC were trypsinized and suspended in conditioned media, and 1 ml containing 2.0 ⫻ 105 cells was layered on the coated insert filters. The cells were then stimulated with ANG II (10⫺7M). The lower chamber had 10% serum containing media. After incubation at 37°C for 24 h, the membranes were removed and washed with PBS, and the noninvading cells on the upper surface were removed with a cotton swab. The cells migrating to the lower surface of the membrane were quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenoltetrazolium bromide assay. To determine the role of IL-18 in ANG II-mediated SMC migration, cells were incubated with ␣IL-18 neutralizing antibodies (␣IL-18 Ab) or IL-18BP-Fc (10 ␮g/ml for 1 h) before ANG II addition. To determine the role of Nox1 in ANG II-induced SMC migration, SMC transfected with Nox1 siRNA (50 nM) for 72 h were used. Cell Proliferation SMC proliferation was analyzed by both [3H]thymidine (TdR) and bromodeoxyuridine incorporation as described before (40). PDGF-BB (10 ng/ml) served as a positive control.

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Cell Death Analysis To determine whether transduction of viral vectors and exposure to pharmacological inhibitors negatively affected cell viability, cell death was analyzed using the Cell Death Detection ELISAPLUS. Cell viability was also tested by trypan blue dye exclusion and microscopic visualization of cell morphology and detachment. Statistical Analysis Comparisons between controls and various treatments were performed by ANOVA with post hoc Dunnett’s t-tests. All assays were performed at least three times, and the error bars in the figures indicate the SE. RESULTS

ANG II Induces Rat Carotid SMC Proliferation and Migration SMC migration and proliferation are two distinct cellular responses, and both phenomena contribute to transplant vasculopathy, postangioplasty restenosis, late vein-graft failure, and atherosclerosis. Atherosclerosis is an inflammatory disease (20), and ANG II, the effector peptide of the renin-angiotensinaldosterone system, contributes to its development and progression (30). Here we show that treatment with ANG II significantly increased primary rat SMC proliferation, as evidenced by increased TdR incorporation, an effect markedly attenuated by the AT1 antagonist losartan but not by the AT2 antagonist PD 123319 (Fig. 1A). Bromodeoxyuridine incorporation confirmed the mitogenic effect of ANG II (data not shown). As a positive control, PDGF-BB potently induced SMC proliferation. Similar to its mitogenic effect, ANG II also induced SMC migration (Fig. 1B), an effect that was also markedly attenuated by losartan. Significantly, knockdown of IL-18 or incubation with IL-18 neutralizing antibodies or IL-18 BP before ANG II addition mitigated both the mitogenic (Fig. 1C; knockdown of IL-18 was confirmed by immunoblotting and is shown in inset) and migratory (Fig. 1D) effects of ANG II. These results indicate that ANG II induces SMC proliferation and migration via AT1/IL-18-dependent signaling (Fig. 1). ANG II Induces IL-18 Expression via AT1 Since ANG II induced SMC proliferation and migration in part via IL-18 (Fig. 1), we next investigated whether ANG II induces IL-18 expression and whether IL-18 by itself exerts mitogenic and migratory effects. Indeed, ANG II induced robust IL-18 mRNA expression in SMC (Northern blot, Fig. 2A; RT-qPCR, Fig. 2B). ANG II-induced IL-18 mRNA expression peaked ⬃2 h and remained elevated throughout the 24 h study period (Fig. 2, A and B), and the AT1 antagonist losartan blunted this response (Fig. 2C). Further, ANG II induced time-dependent IL-18 protein expression (Fig. 2D) and stimulated its secretion in an AT1dependent manner (Fig. 2E). Moreover, confirming our earlier results, IL-18 induced significant proliferation and migration of SMC (data not shown). Together, these results indicate that ANG II induces IL-18 expression in SMC via AT1 (Fig. 2). ANG II-Induced IL-18 Expression Is ROS Dependent Oxidative stress plays a critical role in the development and progression of atherosclerosis (45), and ANG II is a potent inducer of ROS generation. The NADPH oxidases comprise a major source of ROS generation in SMC. Although SMC express both Nox1 and Nox4 at basal conditions, ANG II while inducing


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Fig. 1. ANG II stimulates rat carotid artery smooth muscle cell (SMC) proliferation and migration. A: ANG II stimulates SMC proliferation via AT1. When at 70 – 80% confluency, the complete medium on the SMC was replaced with medium supplemented with 0.5% BSA. After 48 h, quiescent SMC were treated with the AT1 antagonist losartan (10 ␮M in water for 1 h), AT2 antagonist PD 12319 (10 ␮M in DMSO for 1 h) or the solvent control DMSO before ANG II addition (10⫺7 M for 48 h). Incorporation of [3H]thymidine (TdR) into DNA was used as a marker of cell proliferation. PDGF-BB (10 ng/ml for 48 h) served as a positive control. *P ⬍ 0.001 vs. untreated (n ⫽ 12). B: ANG II stimulates SMC migration via AT1. Quiescent SMC in medium containing 0.5% BSA were layered on Matrigel-coated filters. Cells were treated with ANG II (10⫺7 M) for 12 h. Lower chambers contained medium with 10% FCS. Cells migrating to the other side of the membrane were quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenoltetrazolium bromide assay. *P ⬍ 0.001 vs. untreated; †P ⬍ 0.01 vs. ANG II (n ⫽ 12). C and D: ANG II-stimulated SMC proliferation and migration are IL-18 dependent. SMC were transfected with small interfering (si)RNA against IL-18 (50 nM for 72 h) or incubated with IL-18 neutralizing antibodies or IL-18BP-Fc (10 ␮g/ml for 1 h) before ANG II addition. Cell proliferation and migration were analyzed as in A and B, respectively. C and D: *P ⬍ 0.001 vs. untreated; †P ⬍ at least 0.05 vs. ANG II (n ⫽ 12).

Nox1 suppresses Nox4 (10). Therefore, we next investigated whether ANG II-induced IL-18 expression is dependent on Nox1mediated ROS generation. Superoxide generation was measured by two independent methods: the lucigenin-enhanced chemilumi-

nescence assay using whole cells and whole cell homogenates, and a luminol-based chemiluminescence assay (the Diogenes assay) (13). ANG II induced significant generation of superoxide in whole cells, an effect that was markedly attenuated by losartan,

Fig. 2. ANG II induces IL-18 expression. A: time-dependent induction of IL-18 mRNA by ANG II. Quiescent SMC were incubated with ANG II (10⫺7 M). At indicated times, DNA-free total RNA was isolated and analyzed for IL-18 mRNA expression by Northern blotting. 28S rRNA served as loading control (C). A representative of 3 independent experiments is shown. B: ANG II-induced IL-18 mRNA expression was confirmed by RT-quantitative (q)PCR using total RNA isolated in A and TaqMan probes. *P ⬍ 0.01 vs. untreated. C: ANG II induces IL-18 mRNA expression via AT1. Quiescent SMC were incubated with losartan (10 ␮M in water for 1 h) before ANG II addition (10⫺7 M for 2 h). IL-18 mRNA expression was analyzed as in B. *P ⬍ 0.01 vs. untreated; †P ⬍ 0.05 vs. ANG II (n ⫽ 3). D: time-dependent induction of IL-18 protein expression by ANG II. Quiescent SMC treated as in A were analyzed for IL-18 protein expression by immunoblotting using antibodies that specifically detect mature IL-18. A representative of 3 independent experiments is shown. Right: ANG II induces IL-18 protein expression via AT1. Quiescent SMC treated as in C were analyzed for mature IL-18 expression by immunoblotting (n ⫽ 3). E: ANG II-stimulated IL-18 secretion is AT1 dependent. Quiescent SMC were treated as in A, but for 24 h, and secreted IL-18 levels were quantified by an ELISA. *P ⬍ 0.001 vs. untreated; †P ⬍ 0.01 vs. ANG II (n ⫽ 6). AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00231.2012 • www.ajpheart.org


the superoxide scavenger Tiron and overexpression of the cytosolic antioxidant enzyme CuZnSOD (Fig. 3A). Further, the NADPH oxidase inhibitor DPI and Nox1 knockdown blunted ANG II-induced superoxide generation (Fig. 3A; knockdown of Nox1 was confirmed by immunoblotting and is shown in inset). Similar results were obtained when the luminol-based chemiluminescence assay was used to measure superoxide generation (Fig. 3B). Replacing whole cells with whole cell lysates in the lucigeninbased assay also gave similar results (Fig. 3C), indicating that ANG II stimulates superoxide in SMC predominantly through Nox1. Importantly, Tiron, DPI, Nox1 knockdown, or ectopic expression of CuZnSOD each attenuated ANG II-induced expression of mature IL-18 protein (Fig. 3D, left and right). Since the small GTPase Rac1 is a potent activator of Nox1 (32), we next

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examined its role in ANG II-induced superoxide generation. Our results show that forced expression of dominant negative myctagged Rac1 (N17rac1) by adenoviral transduction markedly attenuated ANG II-induced superoxide generation (Fig. 3E; expression of myc was confirmed by immunoblotting as shown in inset) and mature IL-18 protein expression (Fig. 3F). These results indicate that ANG II-induced IL-18 expression is ROS and Nox1 dependent (Fig. 3). ANG II-Induced MMP-9 Expression is ROS and IL-18 Dependent SMC migration is facilitated by degradation of extracellular matrix by the matrix metalloproteinases. Since ANG II induced Fig. 3. ANG II induces IL-18 expression via AT1-, Nox1-, and Rac1-dependent reactive oxygen species (ROS) generation. A: ANG II induces superoxide generation in SMC. SMC loaded with dark-adapted lucigenin (5 ␮M) were incubated with ANG II (10⫺7 M) alone or ANG II with losartan (10 ␮M in water for 1 h), diphenyleneiodonium chloride (DPI; 10 ␮M in DMSO for 30 min), Tiron (5 mM for 1 h), or Tempol (1 mM in DMSO). DMSO served as a solvent control. Studies were also performed using SMC transfected with Nox1 siRNA (50 nM for 72 h) or infected with Ad.CuZnSOD [multiplicity of infection (MOI) 100 for 24 h] before ANG II addition. Nonsilencing siRNA and Ad.GFP served as respective controls. Superoxide (O2·⫺) production was quantified using the lucigeninenhanced chemiluminescence assay. Luminescence was measured for 10 s with a delay of 5 s. After background luminescence was subtracted, results [relative light units (RLU)/second/5 ⫻ 104 cells] were expressed as fold increase from untreated controls. Knockdown of Nox1 was confirmed by immunoblotting (inset). *P ⬍ at least 0.01 vs. untreated; †P ⬍ at least 0.05 vs. ANG II ⫾ respective controls (n ⫽ 12). B: ANG II-induced superoxide generation in SMC was confirmed using the Diogenes reagent. Quiescent SMC were treated as in A, and superoxide production was measured using a whole cell assay with the Diogenes reagent. *P ⬍ 0.001 vs. untreated; †P ⬍ at least 0.05 vs. ANG II ⫾ respective controls (n ⫽ 12). C: ANG II induces superoxide generation in SMC homogenates. Lucigenin-enhanced chemiluminescence assays were performed as described in MATERIALS AND METHODS using SMC homogenates. Reaction mixture contained NADPH (100 ␮M). *P ⬍ 0.001 vs. untreated; †P ⬍ 0.01 vs. ANG II ⫾ respective controls (n ⫽ 12). D: ANG II induces IL-18 protein expression via AT1, Nox1, and ROS. SMC treated as in A, but for 2 h, were analyzed for mature IL-18 protein levels by immunoblotting (right and left; n ⫽ 3). E and F: forced expression of dnRac1 blunts ANG II induced superoxide generation (E) and IL-18 protein expression (F). SMC infected with Ad.dnRac1 (N17rac1; MOI 100 for 24 h) were loaded with lucigenin as in A and were incubated with and without ANG II. Superoxide generation (E) was analyzed as in A. Mature IL-18 protein levels at 2 h were analyzed by immunoblotting (F; n ⫽ 3). E: *P ⬍ 0.001 vs. untreated; †P ⬍ 0.01 vs. ANG II ⫹ Ad.GFP (n ⫽ 12).


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significant migration of SMC (Fig. 1), and as MMP-9 plays a critical role in this process (14, 17, 34), we next investigated whether ANG II induces MMP-9 expression. ANG II was found to be a potent inducer of MMP-9 mRNA expression (Fig. 4A), activation (immunoblotting, Fig. 4A, inset, top), and activity (gelatin zymography, Fig. 4A, inset, bottom). Further, losartan, DPI or Nox1 knockdown each inhibited ANG II induction of MMP-9 mRNA (Fig. 4B, left) and protein (Fig. 4B, right) expression. Importantly, MMP-9 knockdown markedly attenuated ANG II-induced SMC migration (Fig. 4C; knockdown was confirmed by immunoblotting and is shown in inset). Similarly, MMP-2/MMP-9 inhibitor 1 (MMP2/9I) or the broad-spectrum MMP inhibitors Marimastat and GM 6001 (4) inhibited ANG II-induced SMC migration (Fig. 4D). Since ANG II induces IL-18 expression (Fig. 2) and as IL-18 is a potent inducer of MMP-9 expression (4), we next investigated whether MMP-9 induction by ANG II requires IL-18. Indeed, our data show that knockdown of IL-18 or preincubation with ␣IL-18 Ab or IL-18 BP inhibited ANG II-induced MMP-9 expression (Fig. 4E). These results indicate that ANG II is a potent inducer of MMP-9 in SMC, and this effect is mediated in part via ROS, Nox1, and IL-18 (Fig. 4). Physical Association of AT1 with Nox1 We have shown that ANG II induces SMC migration and proliferation via AT1- and Nox1-dependent superoxide gener-

ation (Fig. 3). Previously, we reported that AT1 physically associates with whole Nox2 in primary cardiomyocytes and that ANG II enhanced their binding in vivo (44). Therefore, we investigated whether AT1 also binds Nox1. Using GST pulldown assays, we found that labeled whole Nox1 bound to a GST fusion protein containing the C-terminal region (amino acids 303–359) of AT1 but not to GST itself (Fig. 5A). The Nox proteins are characterized by six transmembranous domains located in the N-terminal region and an extended cytoplasmic C-terminal region that binds both the substrate NADPH and the cofactor FAD. To define the AT1/Nox1 interaction further, and to determine whether this same cytoplasmic C-terminal region may contribute to the binding to AT1, GST pull-down assays were carried out between a fusion protein of maltose binding protein coupled to the cytoplasmic domain of Nox1 [MBPNox1(289 –359)] and GST- AT1(303–359). A strong binding was seen between these two constructs (Fig. 5B). No binding was observed between GST and MBP or MBP-Nox1(289 –359) or between GST- AT1(303–359) and MBP demonstrating the specificity of association in this assay. Thus the C-terminal cytoplasmic domain of Nox1 is responsible for at least part of the interaction with AT1. To determine if Nox1 interacts with AT1 in vivo, we carried out reciprocal immunoprecipitation and immunoblotting of solubilized membrane preparations and whole cell lysates from SMC treated or not with ANG II for 15 min. Our results indicate that while AT1 and Nox1 do associate

Fig. 4. ANG II-induced metalloproteinase (MMP)-9 expression is Nox1 and ROS dependent. A: ANG II induces MMP-9 expression and activity. Quiescent SMC were incubated with ANG II for 2 h (mRNA and protein) and 24 h (activity). MMP-9 mRNA expression was analyzed by RT-qPCR (n ⫽ 6), protein levels by immunoblotting that detect both pro- and mature forms (n ⫽ 3), and activity using culture supernatants and zymography (n ⫽ 3). *P ⬍ 0.001 vs. untreated. B: ANG II induces MMP-9 expression via AT1, Nox1, and ROS. Quiescent SMC treated with losartan (10 ␮M in water for 1 h), DPI (10 ␮M in DMSO for 30 min), or transfected with Nox1 siRNA (50 nM for 72 h) before ANG II addition (10⫺7 M for 2 h). MMP-9 mRNA expression was analyzed by RT-qPCR (left; n ⫽ 6) and protein levels by immunoblotting (right; n ⫽ 3). *P ⬍ 0.001 vs. untreated; †P ⬍ at least 0.05 vs. ANG II ⫹ respective controls. C: knockdown of MMP-9 blunts ANG II induced SMC migration. SMC transfected with MMP-9 siRNA (5 nM for 48 h) were treated with ANG II (10⫺7 M for 12 h) and then analyzed for migration as in Fig. 1B. *P ⬍ 0.001 vs. untreated; †P ⬍ 0.05 vs. ANG II (n ⫽ 12). D: broad-spectrum MMP inhibitors inhibit ANG II induced SMC migration. Quiescent SMC in medium containing 0.5% BSA were layered on Matrigel basement membrane matrix-coated filters. Cells were treated with MMP2/9 inhibitor (1 ␮M in DMSO for 15 min), Marimastat (10 mM in DMSO), or GM 6001 (10 ␮M in DMSO for 15 min) before ANG II addition (10–7M for 12 h). Cell migration was analyzed as in Fig. 1B. *P ⬍ 0.001 vs. untreated; †P ⬍ 0.01 vs. ANG II (n ⫽ 12). E: ANG II induces MMP-9 expression via IL-18. SMC were transfected with IL-18 siRNA (50 nM for 72 h) or incubated with IL-18 neutralizing antibodies or IL-18BP (10 ␮g/ml for 1 h) before ANG II addition. MMP-9 expression was analyzed as in A. *P ⬍ 0.001 vs. untreated; †P ⬍ 0.01 vs. ANG II ⫾ respective controls (n ⫽ 12). AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00231.2012 • www.ajpheart.org


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Fig. 5. ANG II increases the physical association of AT1 and Nox1 in vitro and in vivo. A: Nox1 binds the C-terminal domain of AT1 in vitro. A pull-down assay was carried out between a protein composed of the C-terminal cytoplasmic domain of AT1 fused to GST [GST-AT1(303–359)], prebound to GSH-Sepharose beads, and in vitro transcribed/translated, 35S-methionine-labeled rat Nox1 ([35S]Met-Nox1). GST was used as a binding specificity control. Upper panel, aliquots of labeled Nox1 added to GST or GST-AT1(303–359) (equivalent to 2% of the total, “Input”), and the bound Nox1 eluted with SDS-PAGE treatment buffer from the GSH-Sepharose beads after being washed (equivalent to 10% of eluate, “Bound”) were analyzed by SDS-PAGE and fluorography. Bottom: to show the equal loading of the GSH-Sepharose beads with GST and GST-AT1(303–359), equal volumes of the eluates were analyzed by SDS-PAGE and stained for protein with GelCode Blue. B: C-terminal domain of AT1(303–359) interacts with the C-terminal cytoplasmic domain of Nox1(289 –563). GSH-Sepharose was loaded with either GST or GST-AT1(303–359) as in A, and binding experiments were carried out with in vitro transcribed/translated, [35S]maltose-binding protein (MBP), or a fusion protein of MBP and the C-terminal domain of rat Nox1 {[35S]Met-MBP-Nox1(289 –563)}. Analysis by SDS-PAGE and fluorography were as in A. C: AT1 and Nox1 association in vivo is increased by ANG II. SMC were treated with or without ANG II (10⫺7 M for 15 min), and AT1 and Nox1 binding was analyzed by immunoprecipitation/immunoblot (IP/IB) using solubilized membrane fraction (left and middle) or whole lysates (right). All blots are representatives from three independent experiments. D: AT1/Nox1 binding is reduced in postadsorption supernatants. SMC were treated as in C. Following IP of solubilized membrane fraction, postadsorption supernatants were analyzed for Nox1 (left) or AT1 (right) by IB (n ⫽ 3). E: IL-18 fails to modulate AT1 binding to Nox1. Quiescent SMC were treated with IL-18 (1 ng/ml for 15 min). ANG II served as a positive control. Membrane fraction was analyzed for AT1/Nox1 binding by IP/IB (n ⫽ 3). F: IL-18 fails to modulate ANG II induced AT1/Nox1 binding. Quiescent SMC treated with ANG II (10⫺7 M) or ANG II (10⫺7 M) ⫹ IL-18 (1 ng/ml) for 15 min were analyzed for AT1/Nox1 binding by IP/IB using the membrane fraction (n ⫽ 3).


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in vivo at low levels, this association was increased with ANG II treatment (Fig. 5C, left). Similar results were obtained when the converse immunoprecipitation/immunoblot experiments were carried out (Fig. 5C, middle). Immunoprecipitation/immunoblot using whole cell lysates gave similar results (Fig. 5C, right). To demonstrate the efficiency of the immunoprecipitation step, we performed additional immunoprecipitation/ immunoblot experiments using the post-adsorption supernatants. The results in Fig. 5D show that the postadsorption supernates contained markedly reduced levels of AT1 and Nox1 and therefore reduced AT1/Nox1 binding (AT1 antibodies, left; Nox1 antibodies, right). Thus under the conditions of the experiment, the majority of the cell AT1 and Nox1 was efficiently immunoprecipitated in the first adsorption with antibody. Since IL-18 mediates part of the ANG II effects (Fig. 1), we next examined whether IL-18 regulates AT1 binding to Nox1. Our results show that IL-18 neither changes the basal binding of AT1 with Nox1 (Fig. 5E) nor modulates ANG II-induced increases in AT1/Nox1 binding (Fig. 5F), indicating that although IL-18 mediates part of ANG II signaling, it fails to modulate either AT1 binding to Nox1 or ANG II-induced increases in AT1/Nox1 binding (Fig. 5). ANG II Induces IL-18 and MMP-9 Expression via IKK␤/p65 and JNK/AP-1 We have demonstrated that ANG II induces SMC proliferation and migration via Nox1/ROS/IL-18 (Fig. 1). Since Il18 is an NF-␬B- and AP-1-responsive gene (2, 48), we investigated whether ANG II-induced IL-18 expression is NF-␬B and AP-1 dependent. Our results show that ANG II is a potent inducer of IKK␤ phosphorylation, an effect that was markedly attenuated by losartan, DPI, and Nox1 knockdown (data not shown). Further, ANG II induced timedependent p65 phosphorylation (data not shown). Importantly, ANG II-induced p65 phosphorylation and nuclear translocation are AT1, Nox1, and ROS dependent (Fig. 6A). Moreover, ANG II induced IL-18 and MMP-9 expressions via IKK␤ and p65 (Fig. 6B). Since Il18 and MMP9 are also AP-1-responsive genes (2, 4), we next investigated whether ANG II induces JNK and AP-1 activation. ANG II strongly induced JNK phosphorylation, an effect markedly attenuated by losartan, DPI, and Nox1 knockdown (data not shown). ANG II induced c-Jun phosphorylation and nuclear translocation (data not shown), and importantly, these responses were AT1, Nox1, and ROS dependent (Fig. 6C). Moreover, ANG II induced IL-18 and MMP-9 expressions via JNK and c-Jun (Fig. 6D). Together, these results indicate that ANG II induces IL-18 and MMP-9 expression via Nox1/ROS-dependent IKK␤/NF-␬B and JNK/AP-1 activation (Fig. 6). IL-18 Stimulates SMC Proliferation and Migration via Nox1Dependent ROS Generation and NF-␬B and AP-1 Activation Since ANG II induced SMC migration and proliferation via IL-18 (Fig. 1), we next investigated whether IL-18-induced SMC proliferation and migration are Nox1 and ROS dependent. Initially, we investigated the effect of IL-18 on ROS generation. IL-18 induced significant superoxide generation in SMC, an effect markedly attenuated by DPI and Nox1 knock-

Fig. 6. ANG II activates NF-␬B and AP-1 via AT1, Nox1, and ROS. A: ANG II induces p65 phosphorylation and nuclear translocation. SMC pretreated with losartan (10 ␮M in water for 1 h), DPI (10 ␮M in DMSO for 30 min), or transfected with Nox1 siRNA (50 nM for 72 h) were incubated with ANG II (10⫺7 M for 1 h). Phospho-p65 (Ser536) levels were analyzed by immunoblotting using nuclear protein extracts (n ⫽ 3). B: ANG II induces IL-18 and MMP-9 expression via IKK␤ and p65. SMC infected with Ad.kdIKK␤ or Ad.dnp65 (MOI 100) were treated with ANG II (10⫺7 M for 2 h) and analyzed for mature IL-18 and MMP-9 protein expression by immunoblotting (n ⫽ 3). C: ANG II induces c-Jun phosphorylation and nuclear translocation. SMC treated as in A were analyzed for phospho-c-Jun levels by immunoblotting using nuclear protein extracts (n ⫽ 3). D: ANG II induces IL-18 and MMP-9 expression via JNK and c-Jun. SMC were infected with Ad.dnJNK1 or Ad.dnc-Jun (MOI 100) before ANG II addition (10⫺7 M for 2 h). Mature IL-18 and MMP-9 protein levels were analyzed by immunoblotting (n ⫽ 3).

down (Fig. 7A), and ectopic expression of mutant Rac1 (Fig. 7B). Further, IL-18 induced NF-␬B (Fig. 7C) and AP-1 (Fig. 7D) DNA binding activities via Nox1 and ROS. IL-18 also induced NOX1 transcription in AP-1-dependent manner (data not shown). Notably, IL-18 induced SMC proliferation (Fig. 7E) and migration (Fig. 7F) in a Nox1- and ROS-dependent manner, indicating that IL-18, similar to ANG II, induces superoxide generation, NF-␬B and AP-1 activation, and SMC migration and proliferation, all via Nox1 (Fig. 7). Continuous Infusion of ANG II Enhances Carotid Artery Hyperplasia In Vivo Using histomorphometric analysis and 3H-thymidine labeling, Daemen et al. (7) previously reported that continuous infusion of ANG II for up to 14 days induces SMC proliferation and increases the medial thickness of carotid arteries. Since we demonstrated that ANG II induces significant proliferation and migration of carotid artery SMC in vitro (Fig. 1), we next examined whether ANG II infusion increases carotid artery medial thickness in vivo. To confirm that ANG II infusion exerted biological effects, we first analyzed SBP and myocardial hypertrophy. Corroborating our previously published report (44), continuous infusion of ANG II for 7 days enhanced SBP and cardiac hypertrophy in vivo (data not shown), effects that were markedly attenuated by losartan cotreatment (data not shown). Furthermore, ANG II infusion induced significant hyperplasia in carotid artery, an effect that was markedly attenuated by losartan co-treatment (Fig. 8A). Neither saline nor saline ⫹ losartan modulated SBP or heart weights (data not shown) or induced a hyperplastic response (data not shown). These results indicate that ANG II infusion increases SMC hyperplasia and medial thickness in vivo in an AT1-dependent manner (Fig. 8).



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Fig. 7. IL-18 induces SMC proliferation and migration via Nox1, Rac1, and ROS. A: IL-18 enhanced superoxide generation in SMC is inhibited by DPI and Nox1 knockdown. SMC treated as in Fig. 3A, but with IL-18 (1 ng/ml), were analyzed for superoxide production by the lucigenin-enhanced chemiluminescence assay. *P ⬍ 0.001 vs. untreated; †P ⬍ at least 0.05 vs. IL-18 ⫾ respective controls (n ⫽ 12). B: IL-18 enhanced superoxide generation is Rac1 dependent. SMC were infected with Ad.N17Rac1 as in Fig. 3E before IL-18 addition. Superoxide production was analyzed by the lucigenin-enhanced chemiluminescence assay. *P ⬍ 0.01 vs. untreated; †P ⬍ 0.05 vs. IL-18 ⫾ Ad.GFP (n ⫽ 12). C: IL-18 activates NF-␬B via Nox1 and ROS. SMC treated as in A, but for 1 h, were analyzed for NF-␬B DNA binding activity by EMSA using nuclear protein extracts and MMP9 gene-specific primers (n ⫽ 3). Specificity of DNA binding, indicated by an arrow, was verified in competition experiments. Unincorporated labeled probe that runs to the bottom of gel is not shown. Lamin A/C (nuclear) and GAPDH (cytoplasmic) served as loading and purity controls and are shown at bottom. D: IL-18 activates AP-1 via Nox1 and ROS. SMC treated as in A, but for 1 h, were analyzed for AP-1 DNA binding activity by EMSA using nuclear protein extracts and MMP9 gene-specific primers (n ⫽ 3). E and F: IL-18stimulated SMC proliferation and migration are dependent on Nox1. SMC transfected with Nox1 siRNA (50 nM for 72 h) were stimulated with IL-18 (1 ng/ml) and assayed for proliferation and migration as detailed in Fig 1. *P ⬍ 0.001 vs. untreated; †P ⬍ 0.01 vs. IL-18 (n ⫽ 12).

ANG II Infusion Is Associated with Increased ROS Generation, Nox1 mRNA Expression, and AT1 Binding to Nox1 In Vivo in Endothelium-Denuded Carotid Artery

these results indicate that ANG II infusion is associated with increased levels of phospho-IKK␤, -p65, -JNK, and -c-Jun in the endothelium-denuded carotid artery (Fig. 9).

We next examined whether ANG II infusion increases ROS generation in vivo. Confirming our in vitro results (Fig. 3A), ANG II infusion enhanced superoxide generation in endothelium-denuded rat carotid artery homogenates, an effect markedly attenuated by DPI (Fig. 8B). Further, ANG II infusion increased Nox1 mRNA expression in an AT1-dependent manner (Fig. 8C). Notably, immunoprecipitation/immunoblot showed AT1 physical association with Nox1 in vivo in endothelium-denuded carotid artery extracts from saline-infused animals, and their binding was enhanced following ANG II infusion (Fig. 8D, top), despite the fact that overall AT1 expression was reduced in the ANG II infused animals (Fig. 8D, bottom). These results indicate that ANG II infusion is associated with increased DPI and AT1-dependent ROS generation, Nox1 mRNA expression, and Nox1 physical association with AT1 in vivo (Fig. 8).

ANG II Infusion Is Associated with Increased IL-18 and MMP-9 Expression in Endothelium-Denuded Carotid Artery

ANG II Infusion Is Associated with Increased Phosphorylated Levels of IKK, JNK, p65, and c-Jun in Endothelium-Denuded Carotid Artery Similar to its effects in SMC in vitro (Fig. 6), ANG II infusion increased levels of p-IKK␤, p-p65, p-JNK and p-c-Jun in the endothelium-denuded carotid arteries (Fig. 9A; densitometric analyses are shown in B), and these levels were markedly reduced in the losartan co-treatment animals (Fig. 9C; densitometric analyses are shown in D). Neither saline nor saline ⫹ losartan modulated the levels of total and phosphorylated IKK␤, JNK, p65, and c-Jun (data not shown). Together,

Since ANG II infusion is associated with increased levels of NF-␬B/p65 and AP-1/c-Jun (Fig. 6), we next investigated whether its infusion enhances expression levels of NF-␬B- and AP-1-responsive IL-18 and MMP-9 in vivo in carotid arteries. Immunoblotting showed increased expression of mature 18kDa IL-18 in endothelium-denuded carotid artery from ANG II-infused animals in vivo (Fig. 10A), and this increase was significantly attenuated in losartan cotreated animals (Fig. 10B). Similarly, ANG II infusion enhanced both pro- and active forms of MMP-9 (Fig. 10C), and these effects were also markedly reduced in the losartan cotreated animals (Fig. 10D). Neither saline nor saline ⫹ losartan modulated IL-18 and MMP-9 levels (data not shown). These results indicate that ANG II infusion enhances both IL-18 and MMP-9 in endothelium-denuded carotid arteries in an AT1-dependent manner (Fig. 10). DISCUSSION

The octapeptide ANG II can play a causal role in the development and progression of atherosclerosis and restenosis, along with the pathobiological contribution of oxidative stress and inflammation (11, 15, 20, 30, 45). SMC migration and proliferation are two biologically distinct phenomena, both of which contribute to medial thickness resulting in stenosis and associated pathologies. Here we show that ANG II is a potent


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Fig. 8. ANG II infusion induces hyperplasia, oxidative stress, and AT1/Nox1 binding in carotid artery. A: ANG II infusion induces carotid artery hyperplasia. Male SD rats were infused with ANG II (700 ␮g·kg⫺1·day⫺1) via miniosmotic pumps for 7 days. Saline served as a control. A subgroup of animals infused with ANG II was cotreated with losartan, a selective AT1 blocker, via drinking water (400 mg/l; 40 mg·kg body wt⫺1·day⫺1). The right carotid arteries were fixed, embedded in paraffin, sectioned, and stained by hematoxylin and eosin. Intimal (I) and medial (M) thickness was quantified by computerized digital microscopic software. Representative hematoxylin and eosin-stained carotid artery sections are shown. Quantitation is shown at right. *P ⬍ 0.05 vs. ANG II (n ⫽ 6/group). B: ANG II induces DPI-inhibitable superoxide generation. Male Sprague-Dawley rats were treated as in A. NADPH-dependent superoxide production was measured using the lucigenin-enhanced chemiluminescence assay and endothelium-denuded carotid artery homogenates from the indicated groups. Experiments were also performed in the presence of DPI (10 ␮M in DMSO) using carotid artery homogenates from ANG II-infused animals. *P ⬍ 0.001 vs. saline; †P ⬍ at least 0.05 vs. ANG II or ANG II ⫹ DMSO (n ⫽ 6/group). C: losartan attenuates ANG II-induced Nox1 mRNA expression in vivo. Nox1 mRNA expression in endothelium-denuded carotid arteries was analyzed by RT-qPCR. *P ⬍ 0.001 vs. untreated; †P ⬍ 0.01 vs. ANG II (n ⫽ 6/group). D: ANG II infusion is associated with enhanced AT1/Nox1 binding in vivo in carotid arteries. Binding of AT1 and Nox1 in vivo is increased by ANG II. Carotid artery homogenates from saline and ANG II-infused animals was analyzed for AT1/Nox1 binding by IP/IB. Blots are representatives from 3 independent experiments.

inducer of oxidative stress in rat carotid artery SMC, and the AT1 antagonist losartan, the flavoprotein inhibitor DPI, and siRNA-mediated Nox1 knockdown can each blunt this effect. Here we show for the first time that AT1 physically associates with Nox1 in vitro and in vivo and ANG II increases their interaction in vivo. Further, ANG II induces IL-18, a mitogenic and proinflammatory cytokine (4, 40), via AT1/Nox1/ROSmediated IKK␤/p65 and JNK/c-Jun activation, and ANG IIinduced SMC migration and proliferation are IL-18 dependent. ANG II also induced MMP-9 expression in SMC via NF-␬B

and AP-1 and Nox1 via AP-1. Similar to ANG II, IL-18 induces ROS generation, NF-␬B and AP-1 activation, and SMC migration and proliferation in a DP1- and Nox1-dependent manner. Of note, IL-18 induces NOX1 transcription via AP-1. Recapitulating these in vitro effects on SMC, continuous infusion of ANG II for 7 days was associated with 1) increased ROS generation; 2) Nox1 induction; 3) increased AT1 binding to Nox1; 4) increased levels of phospho-IKK␤, -p65, -JNK, and -c-Jun; 5) upregulation of IL-18 and MMP-9 expression; and 6) increased carotid artery medial thickness, all in an

Fig. 9. ANG II infusion is associated with increased levels of phospho-IKK␤, -p65, -JNK, and -c-Jun in endotheliumdenuded carotid arteries. A: ANG II infusion increases levels of activated IKK␤, JNK, p65, and c-Jun in endothelium-denuded carotid artery homogenates. Expression levels of indicated proteins (total and phosphorylated) were analyzed by immunoblotting using cleared carotid artery homogenates from saline and ANG II-infused animals. The intensity of immunoreactive bands was semiquantified by densitometry, and the results are summarized in B. *P ⬍ 0.05 vs. saline (n ⫽ 4/group selected at random). C: losartan coadministration blunts ANG II induced phosphoIKK␤, -p65, -JNK, and -c-Jun levels in endotheliumdenuded carotid arteries. Expression levels of indicated proteins in endothelium-denuded carotid arteries from ANG II and ANG II ⫹ losartan-treated animals were analyzed by immunoblotting as in A. Intensity of immunoreactive bands was semiquantified by densitometry, and the results are summarized in D. *P ⬍ 0.05 vs. ANG II (n ⫽ 3/group selected at random).



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Fig. 10. ANG II infusion is associated with increased levels of IL-18 and MMP-9 in endothelium-denuded carotid arteries. A: ANG II infusion increases IL-18 expression. Expression levels of mature IL-18 levels were analyzed by immunoblotting using cleared carotid artery homogenates from saline and ANG II-infused animals (n ⫽ 4/group selected at random). B: losartan coadministration blunts ANG II-induced IL-18 expression. Expression levels of mature IL-18 in endothelium-denuded carotid arteries from ANG II and ANG II ⫹ losartan-administered animals were analyzed by immunoblotting (n ⫽ 3/group selected at random). C: ANG II infusion increases MMP-9 expression. Expression levels of pro- and active MMP-9 were analyzed by immunoblotting using cleared carotid artery homogenates from saline and ANG II-infused animals (n ⫽ 4/group selected at random). D: losartan coadministration blunts ANG II-induced MMP-9 expression. Expression levels of MMP-9 in endothelium-denuded carotid arteries from ANG II and ANG II ⫹ losartan-administered animals were analyzed by immunoblotting (n ⫽ 3/group selected at random). E: schema demonstrating the signal transduction pathway(s) involved in ANG II-mediated SMC hyperplasia. ANG II stimulates SMC migration and proliferation via AT1/Nox1-dependent IL-18 induction, and this process may involve the physical association of AT1/Nox1 (solid blue arrow). Induced IL-18, via IL-18R, amplifies the ANG II-induced, redox-dependent inflammatory cascades, by activating similar promitogenic and promigratory signal transduction pathways. The AT1/Nox1/IL-18 pathway may be critical in hyperplastic vascular diseases, including atherosclerosis and restenosis.

AT1-dependent manner. These results indicate that the AT1/ Nox1/IL-18 signaling may be a critical mediator of ANG II-induced atherogenesis and restenosis, and thus IL-18 may be a potential therapeutic target in these inflammation-mediated hyperplastic processes (Fig. 10E). Although it is well established that ANG II can induce cardiomyocyte hypertrophy both in vitro and in vivo (3, 27, 38, 50), its effects on SMC hypertrophy and proliferation are variable. Here we show that ANG II induced a marked increase in both migration and proliferation of SMC, but not hypertrophy (data not shown). Further, we also show that ANG II infusion increases carotid artery medial thickness in vivo, due in part to SMC hyperplasia. This hyperplastic effect of ANG II is consistent with a recently published study (37), demonstrating that ANG II infusion increased aortic medial thickness. However, the type of ANG II-induced medial thickening was location dependent, as ANG II induced hyperplasia of SMC in the ascending aorta but hypertrophy in the descending and other parts of aorta. These differential effects were attributed to the various embryonic origins of SMC. The SMC in the ascending aorta are of neural crest origin, whereas the SMC in thoracic and abdominal aorta are of somite and splanchnic mesoderm lineages, respectively (26). Similar to ascending aorta, the SMC from carotid artery are of neural crest origin and therefore exhibited migration and proliferation in response to ANG II in our studies. ANG II is a potent inducer of oxidative stress. In SMC, NADPH oxidases play a critical role in ANG II-mediated ROS generation. Among the Nox isoforms, SMC predominantly express Nox1 and Nox4 under basal conditions. Interestingly, ANG II has been shown to suppress Nox4 but activate Nox1 (10). Here, we show that ANG II stimulates significant ROS generation in SMC, an effect that was markedly attenuated by DPI, Nox1 knockdown, Tiron, and overexpression of the cytosolic CuZnSOD. Similarly, the AT1 antagonist losartan attenuated ANG II-induced ROS generation, indicating that ANG II-induced ROS generation to be AT1 and Nox1 dependent.

Using GST pull-down assays and reciprocal immunoprecipitation and immunoblotting, we further show that AT1 physically associates with Nox1 both in vitro and in vivo, and ANG II treatment increased their binding in vivo. The increased binding between Nox1 and GST-AT1(303–359) compared with that seen for GST alone strongly suggests that the C-terminal amino acids of AT1 contribute to the binding of Nox1. Further, by in vitro studies, we also showed that this region of the AT1 receptor protein interacts with the cytoplasmic C-terminal domain of Nox1, the region that also binds the Nox substrate NADPH and the cofactor FAD. These results on Nox1-AT1 interaction are novel and significant, complementing our findings of ANG II-enhanced binding of Nox2 to AT1 in primary mouse cardiomyocytes (44). Overall, it appears that, despite the cell-type specificity of Nox isoform expression, AT1 binding to NADPH oxidases is a general phenomenon. Of note, the binding of ANG II to AT1 has also been shown to enhance the interaction of the receptor with caveolin-1, a major caveolar structural protein, and promote its trafficking into caveolae/ lipid rafts that in turn promotes Rac1 translocation and increased local ROS production (18, 52). In fact, ANG IIinduced ROS generation was markedly attenuated following forced expression of the dominant-negative mutant of Rac1 (N17rac1). Since Nox1 and Rac1 are present in lipid rafts, it is reasonable to speculate that the colocalization of AT1, Nox1, and Rac1 in these membrane domains may increase their interaction and enhance ROS production. In studies of restenosis, a hyperplastic vascular disease, Szocs et al. (46) have demonstrated superoxide generation in rat carotid artery 3–15 days following balloon injury. They further demonstrated that the ROS were not produced by infiltrating inflammatory cells but rather by medial and neointimal smooth muscle cells and adventitial fibroblasts (46). They also showed upregulation of Nox1 and p22phox mRNA expression at day 3 and its persistence for up to 14 days after injury. Interestingly, Nox4 expression was increased moderately and in a delayed manner (15 days; Ref. 46). Consistent with those studies, and recapitulating our in vitro findings, here


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we show that continuous infusion of ANG II for 7 days was associated with increased Nox1 mRNA expression and DPIinhibitable Nox1-dependent ROS generation in endotheliumdenuded carotid artery. Further, we show that ANG II infusion increased phospho-IKK␤, -JNK, -p65, and -c-Jun levels; induced IL-18 expression; and increased carotid artery medial thickness. Significantly, all these effects were attenuated by cotreatment with the AT1 antagonist losartan. These results indicate that Nox1-mediated ROS generation activates two critical cellular signal transduction pathways that result in NF-␬B and AP-1 activation and IL-18 induction. These results also support the notion that Nox1 is a key factor in medial hyperplasia, independent of the stimulus (balloon injury vs. ANG II infusion). The role of Nox1 in SMC migration has also been demonstrated in a thrombin-induced, EGFR-dependent model in SMC isolated from thoracic aorta (19). Although crosstalk between AT1 and EGFR has been reported, Takaguri et al. (47) recently demonstrated that ectopic expression of caveolin-1 inhibited ANG II-induced EGFR ligand shedding, EGFR transactivation, ERK activation, and SMC hypertrophy and migration. Since ANG II induces ROS generation, and AT1, Nox1, and Rac1 colocalize in caveolae and lipid rafts (18, 52), and caveolin-1 blunts ANG II-mediated EGFR transactivation (47), it is possible that ANG II-mediated carotid artery SMC migration and proliferation are mainly due to ANG II/AT1/Nox1 signaling rather than EGFR and that ANG II/EGFR crosstalk may be cell type- and stimulus-specific. Studies are in progress to confirm this hypothesis. Notably, Nox1 has also been shown to contribute to SMC migration induced by PDGF or bFGF (21, 42). Together, these results indicate Nox1-mediated ROS generation is critical in both SMC migration and proliferation. In addition to their involvement in inflammation and injury, ROS act as second messengers to regulate multiple signal transduction pathways, including activation of the redox-sensitive transcription factors NF-␬B (12) and AP-1. Here we report that ANG II induces phosphorylation of IKK␤ at Ser176/180 and that Nox1 knockdown, or forced expression of mutant IKK␤, each markedly attenuated ANG II induced p65 phosphorylation and nuclear translocation, NF-␬B DNA binding activity, IL-18 and MMP-9 induction, and SMC migration and proliferation. Our results also demonstrate that ANG II induces phosphorylation and activation of the mitogen-activated protein kinase JNK in SMC via AT1/Nox1/ROS signaling. Similar results were reported recently in bFGF-induced SMC migration (42). In that study, bFGF induced JNK phosphorylation via Nox1, and inhibition of Nox1 and JNK blunted SMC migration. Interestingly, the small GTPase Rac1 has also been shown to selectively activate JNK and c-Jun transcriptional activity (31). Here we show that ANG II induces c-Jun phosphorylation and nuclear translocation, AP-1 DNA binding activity, IL-18 and MMP-9 induction, and SMC migration and proliferation via Nox1 and JNK. Of note, NOX1 is an AP-1 responsive gene (29), and ANG II induced its transcription via AP-1 in SMC (data not shown). Our results also suggest that in addition to being a NF-␬Band AP-1-responsive gene, IL-18 is also a potent inducer of these two oxidative stress-responsive transcription factors in SMC and thereby sustains inflammatory signaling. Similar to ANG II, IL-18 induced ROS generation via Nox1, induced NOX1 transcription via AP-1, activated both NF-␬B and AP-1,

and mediated ANG II-induced MMP-9 expression. Interestingly, while it mediated several ANG II-induced downstream signaling pathways, it failed to modulate either basal AT1/ Nox1 binding or further enhanced ANG II-mediated increases in AT1/Nox1 association, indicating that IL-18 may synergize with ANG II in activating similar distal signal transduction pathways that ultimately lead to NF-␬B- and AP-1-dependent SMC migration and proliferation. A crosstalk has also been reported between NF-␬B and AP-1 in the transcriptional regulation of target genes. Interestingly, NF-␬B positively regulates JNK activity in a cell- and stimulus-specific manner (1, 23). Since ANG II and IL-18 both activate NF-␬B and AP-1, and since IL-18, MMP-9, and several other inflammatory cytokines are NF-␬B and AP-1 responsive, it is critical to identify and target an upstream kinase or adaptor molecule to blunt ANG II/IL-18-mediated NF-␬B and AP-1 activation, proinflammatory signaling, and SMC migration and proliferation. In summary, our results strongly suggest that the AT1/Nox1/IL-18 pathway is a critical factor, and thus a potential therapeutic target, in hyperplastic vascular diseases. ACKNOWLEDGMENTS The contents of this report do not represent the views of the Department of Veterans Affairs or the United States Government. GRANTS This work was supported by the Veterans Affairs Office of Research and Development–Biomedical Laboratory Research and Development Service Award 1IO1BX000246 and National Heart, Lung, and Blood Institute Grant HL-86787 to BC. P. Delafontaine is supported by National Heart, Lung, and Blood Institute Grants HL-70241 and HL-80682. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: A.J.V., T.Y., S.N.M., S.S.S., M.K., and B.C. performed experiments; A.J.V., T.Y., S.N.M., S.S.S., M.K., P.D., and B.C. analyzed data; A.J.V., T.Y., S.N.M., M.K., R.A.C., and B.C. interpreted results of experiments; A.J.V., S.S.S., M.K., and B.C. prepared figures; A.J.V., R.A.C., and P.D. edited and revised manuscript; A.J.V., T.Y., S.N.M., S.S.S., M.K., R.A.C., P.D., and B.C. approved final version of manuscript; S.N.M., R.A.C., P.D., and B.C. conception and design of research; B.C. drafted manuscript. REFERENCES 1. Bubici C, Papa S, Dean K, Franzoso G. Mutual cross-talk between reactive oxygen species and nuclear factor-kappa B: molecular basis and biological significance. Oncogene 25: 6731–6748, 2006. 2. Chandrasekar B, Marelli-Berg FM, Tone M, Bysani S, Prabhu SD, Murray DR. Beta-adrenergic stimulation induces interleukin-18 expression via beta2-AR, PI3K, Akt, IKK, and NF-kappaB. Biochem Biophys Res Commun 319: 304 –311, 2004. 3. Chandrasekar B, Mummidi S, Claycomb WC, Mestril R, Nemer M. Interleukin-18 is a pro-hypertrophic cytokine that acts through a phosphatidylinositol 3-kinase-phosphoinositide-dependent kinase-1-Akt-GATA4 signaling pathway in cardiomyocytes. J Biol Chem 280: 4553–4567, 2005. 4. Chandrasekar B, Mummidi S, Mahimainathan L, Patel DN, Bailey SR, Imam SZ, Greene WC, Valente AJ. Interleukin-18-induced human coronary artery smooth muscle cell migration is dependent on NF-kappaBand AP-1-mediated matrix metalloproteinase-9 expression and is inhibited by atorvastatin. J Biol Chem 281: 15099 –15109, 2006. 5. Chandrasekar B, Vemula K, Surabhi RM, Li-Weber M, OwenSchaub LB, Jensen LE, Mummidi S. Activation of intrinsic and extrinsic proapoptotic signaling pathways in interleukin-18-mediated human cardiac endothelial cell death. J Biol Chem 279: 20221–20233, 2004.


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