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Cardiovascular Research 2007 74(1):169-179; doi:10.1016/j.cardiores.2007.01.017
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Copyright © 2007, European Society of Cardiology

Butylated hydroxyanisole stimulates heme oxygenase-1 gene expression and inhibits neointima formation in rat arteries

Xiao-ming Liua,b, Mohammed A. Azama,b, Kelly J. Peytona,b, Diana Ensenatb, Amit N. Keswanib, Hong Wangc and William Durantea,b,*

aDepartment of Medical Pharmacology and Physiology, University of Missouri, M409 Medical Sciences Building, One Hospital Drive, Columbia, MO 65212, United States
bDepartment of Medicine, Baylor College of Medicine, Houston, TX 77030, United States
cDepartment of Pharmacology, Temple University, Philadelphia, PA 191140, United States

* Corresponding author. Department of Medical Pharmacology and Physiology, University of Missouri, M409 Medical Sciences Building, One Hospital Drive, Columbia, MO 65212, United States. Tel.: +1 573 882 3886; fax: +1 573 884 4276. Email address: durantew{at}health.missouri.edu

Received 25 September 2006; revised 19 January 2007; accepted 23 January 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Butylated hydroxyanisole (BHA) is a synthetic phenolic compound that is a potent inducer of phase II genes. Since heme oxygenase-1 (HO-1) is a vasoprotective protein that is upregulated by phase II inducers, the present study examined the effects of BHA on HO-1 gene expression and vascular smooth muscle cell proliferation.

Methods: The regulation of HO-1 gene expression and vascular cell growth by BHA was studied in cultured rat aortic smooth muscle cells and in balloon injured rat carotid arteries.

Results: Treatment of cultured smooth muscle cells with BHA stimulated the expression of HO-1 protein, mRNA and promoter activity in a time- and concentration-dependent manner. BHA-mediated HO-1 expression was dependent on the activation of NF-E2-related factor-2 by p38 mitogen-activated protein kinase. BHA also inhibited cell cycle progression and DNA synthesis in an HO-1-dependent manner. In addition, the local perivascular delivery of BHA immediately after arterial injury of rat carotid arteries induced HO-1 protein expression and markedly attenuated neointima formation.

Conclusions: These studies demonstrate that BHA stimulates HO-1 gene expression in vascular smooth muscle cells, and that the induction of HO-1 contributes to the antiproliferative actions of this phenolic antioxidant. BHA represents a potentially novel therapeutic agent in treating or preventing vasculoproliferative disease.

KEYWORDS Smooth muscle; Arteries; Gene expression; Restenosis; Angioplasty


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Vascular smooth muscle cell (SMC) proliferation contributes to the development of atherosclerosis and is the central pathophysiological mechanism responsible for the failure of interventional therapeutic approaches to treat occlusive vascular disorders, such as vein bypass graft failure, transplant atherosclerosis, and postangioplasty restenosis [1–3]. Considerable effort has been devoted in designing drugs to target vascular SMC activation and proliferation. However, effective therapy to prevent occlusive vascular disease has not yet been fully established. Recent studies from our laboratory and others indicate that heme oxygenase-1 (HO-1), which catalyzes the degradation of heme to biliverdin, iron and carbon monoxide, is a critical regulator of vascular remodeling [4]. Overexpression of HO-1 blocks vascular SMC proliferation whereas inhibition of HO-1 promotes the mitogenic potential of SMC [5–8]. Furthermore, several studies have reported that induction of HO-1 by hemin attenuates neointima formation following balloon injury of rat carotid arteries [8–10]. Similarly, localized adenovirus-mediated HO-1 gene delivery immediately following arterial injury ameliorates neointima formation in rat carotid and pig femoral arteries [6,11]. In addition, HO-1 deficient mice display enhanced neointima formation following wire induced arterial injury and robust vascular SMC proliferation in a murine vein graft model [6,12]. Moreover, it appears that HO-1 may govern the vascular response to injury in humans since an HO-1 promoter polymorphism linked with impaired inducibility is associated with enhanced restenosis in patients undergoing percutaneous transluminal angioplasty in femoropopliteal arteries or stenting of coronary arteries [13,14].

Butylated hydroxyanisole (BHA) is a synthetic phenolic antioxidant that is primarily utilized as a food preservative. In addition to inhibiting lipid peroxidation, BHA exhibits a wide spectrum of biological activity. Dietary administration of BHA protects against injury from radiation and various toxic compounds [15]. In addition, BHA inhibits carcinogenesis in numerous tissues, including the liver, colon, lung, and breast [15]. These protective properties of BHA are attributed, in part, to its ability to induce phase II detoxifying enzymes such as epoxide hydrolases, glutathione S-transferase, UDP-glucuronosyltransferases, and quinone reductases that results in the metabolic detoxification of carcinogens [16,17]. Furthermore, BHA inhibits the growth of tumor cells and fibroblasts and protects against the development of bronchioloalveolar hyperplastic lesions during pulmonary fibrosis [18–21]. Since HO-1 is an important vasoprotective protein that is upregulated by phase II inducers, the present study examined the effect of BHA on HO-1 gene expression and vascular SMC proliferation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Materials
BHA, collagenase, elastase, SDS, Tris, Tes, Hepes, actinomycin D, EDTA, streptomycin, and minimum essential medium were from Sigma Chemical (St. Louis, MO); tin protoporphyrin-IX was from Frontier Scientific (Logan, UT); LY294002, SB203580, SP600125, and PD98059 were from Calbiochem (La Jolla, CA); a polyclonal HO-1 antibody was from StressGen Biotechnologies (Victoria, Canada); antibodies against NF-E2-related factor 2 (Nrf2), and β-actin were from Santa Cruz Biotechnologies (Santa Cruz, CA); antibodies against phospho-ERK1/2, phospho-JNK1/2, phospho-p38 mitogen-activated protein kinase (MAPK), or phospho-Akt were from Cell Signaling (Beverley, MA); antibody against CD45 was from BD Pharmingen (San Diego, CA); {gamma}-[32P]ATP (3000 Ci/mmol) and [3H]thymidine (90 Ci/mmol) was from NEN-Dupont (Boston, MA); [32P]UTP (400 Ci/mmol) was from Amersham (Arlington Heights, IL).

2.2 Cell culture
Vascular SMC was isolated by enzymatic digestion of rat thoracic aorta and characterized by morphological and immunological criteria [22]. Cells (passages 6 to 10) were serially cultured in minimum essential medium containing 10% bovine serum, 5 mM Tes, 5 mM Hepes, and 100 U/ml streptomycin.

2.3 HO-1 mRNA analysis
Total RNA (30 µg) was loaded onto 1.2% agarose gels, fractionated by electrophoresis, and blot transferred to Gene Screen Plus membranes. Membranes were prehybridized in rapid hybridization buffer and then incubated overnight at 68 °C in hybridization buffer containing [32P]DNA probes (1x108 cpm) for HO-1, GAPDH, or 18S mRNA. DNA probes were generated by RT-PCR and labeled with [32P]dCTP using a random priming kit (Amersham) [7,23]. Following hybridization, membranes were washed and exposed to X-ray film at –70 °C.

2.4 HO-1 promoter analysis
HO-1 promoter activity was determined in promoter/luciferase constructs (1 µg/ml) containing the wild type enhancer (E1) coupled to a minimum HO-1 promoter or the mutant E1 enhancer (M739) that had its three antioxidant responsive element (ARE) core sequences mutated [24]. These promoter constructs, pCMVβ-galactosidase (1 µg/ml), and a plasmid expressing a dominant-negative Nrf2 (dnNrf2; 1 µg/ml) that had its transactivation domain deleted [24] were transfected into SMC using lipofectamine, and cells were exposed to BHA 24 h later. Cells were then collected, lysed, and luciferase activity measured using a luciferase assay system (Promega, Madison, WI) and a TD-Zoe luminometer (Turner Designs Inc., Mountain View, CA). Luciferase activity was normalized with respect to β-galactosidase activity, and expressed as fold induction over control cells. All constructs were generously provided by Dr. Jawed Alam at the Ochsner Clinic Foundation, New Orleans, LA.

2.5 Protein analysis
Vascular SMC were lysed in sample buffer (125 mM Tris [pH 6.8], 12.5% glycerol, 2% SDS, 50 mM sodium fluoride, and trace bromophenol blue) and proteins separated by SDS-PAGE. Following transfer to nitrocellulose membrane, blots were blocked with PBS and nonfat milk (5%) and then incubated with antibodies directed against HO-1 (1:500), Nrf2 (1:100), β-actin (1:200), phospho-ERK1/2 (1:1000), phospho-JNK1/2 (1:1000), phospho-p38 MAPK (1:500), or phospho-Akt (1:1000). Membranes were then washed in PBS, incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-goat antibody and developed with commercial chemoluminescence reagents (Amersham, Arlington Heights, IL).

2.6 Vascular SMC proliferation
The proliferative status of SMC was assessed by monitoring cell cycle progression and DNA synthesis. For cell cycle analysis, SMC were incubated in serum-free media for 48 h prior to treatment with serum (5%) in the presence and absence of BHA for 24 h. Cells were then stained with propidium iodide and DNA fluorescence measured in Dickinson FACScan flow cytometer (Franklin Lakes, NJ). For DNA synthesis, quiescent cells were incubated with serum (5%) in the presence or absence of BHA for 20 h and then [3H]thymidine (1 µCi/ml) was added, and cells incubated for an additional 4 h. SMC were then washed three times with ice-cold PBS, fixed with 10% trichloroacetic acid, and DNA extracted with 0.2% SDS/0.2 N NaOH. [3H]Thymidine incorporation was determined by scintillation spectrophotometry.

2.7 Cell viability
Cell viability was assessed by measuring the uptake of membrane-impermeable stains, propidium iodide and trypan blue, as previously described [25].

2.8 Animals studies
Male Sprague Dawley rats (400–450 g; Charles River Laboratories, Wilmington, MA) were anesthetized (ketamine, xylazine, and acepromazine; 0.7 ml/kg, im; VetMed Drugs, Houston, TX) and the left common carotid artery injured with a Fogarty 2F embolectomy catheter (Baxter Healthcare Corp., Houston, TX), as we previously described [10,11,26]. Immediately after balloon injury, a local polymer based delivery system was used to dispense BHA to the injured vessel wall. The delivery system consisted of 200 µl of a 25% copolymer gel (PLF127; BASF Corporation, Florham Park, NJ) containing BHA (1 mg) that was administered in a circumferential manner to the exposed adventitia of the carotid artery. A separate cohort of animals received an empty gel which has previously been shown to have no effect on vascular remodeling [11,27,28]. PFL127 gels act as a rate-controlling barrier, serving as a vehicle for sustained release of drug; releasing an accumulative amount of drug over a 3-hour period of ~5.5% from a 25% PLF127 gel [29]. Based on this finding, we estimated a delivery dose of 55 µg after 3 h. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.9 Histology
Two weeks after injury, rats were euthanized and vessels collected for protein analysis or the vasculature perfusion-fixed with 10% buffered formalin. The common carotid artery was excised and paraffin-embedded, and 5 µm sections were stained with Verhoff's-Van-Gieson for measurement of vessel dimensions. Microscopic determination of vessel dimensions was performed using Image Plus (Media Cybernetics) and Adobe Photoshop software linked through a digital camera (Leaf Microlumina; Leaf Systems, USA) to a Zeiss Axioskop 50 light microscope (Carl Zeiss, Germany), as we have previously described [10,11,26]. Apoptosis was also monitored by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeled (TUNEL) staining [11]. Leukocyte infiltration was assessed by immunohistochemistry using an antibody against CD45.

2.10 Statistics
Results are expressed as the means±SEM. Statistical differences between groups were evaluated with a Student's two-tailed t-test or by ANOVA with post-hoc Bonferroni's t-test when multiple groups are compared. p values<0.05 were considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Treatment of vascular SMC with BHA for 24 h stimulated a concentration-dependent increase in HO-1 mRNA and protein (Fig. 1A). A dramatic increase in HO-1 expression was noted with 300 µM of BHA. BHA, at concentrations up to 300 µM, had no adverse effect on cell viability; however, higher concentrations of BHA evoked a progressive decline in cell survival. A significant increase in cell death (14.8± 2.6%, p<0.005, n=3) was first noted when cells were exposed to 500 µM of BHA. Incubation of SMC with 1 mM of BHA caused a dramatic toxic response with a 95.8±2.2% (p<0.005, n=3) loss in cell viability. In subsequent experiments, BHA exposure was restricted to non-toxic concentrations. The induction of HO-1 by BHA was delayed, with significant increases in HO-1 mRNA and protein appearing 4 and 8 h, respectively, after BHA administration (Fig. 1B). Incubation of vascular SMC with the transcriptional inhibitor, actinomycin D, completely blocked the induction of HO-1 mRNA and protein by BHA. In contrast, the protein synthesis inhibitor, cycloheximide, only partially inhibited the increase in HO-1 mRNA while totally suppressing the rise in HO-1 protein by BHA. In the absence of BHA, cycloheximide or actinomycin had no significant effect on HO-1 mRNA expression (Fig. 1C). These findings suggest that BHA may directly stimulate HO-1 gene transcription.


Figure 1
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  		Fig. 1 BHA stimulates HO-1 mRNA and protein expression in vascular SMC. A. SMC were treated with BHA (0–300 µM) for 24 h, and HO-1 mRNA and protein determined by northern and western blotting, respectively. B. SMC were incubated with BHA (300 µM) for various times (0–24 h), and HO-1 mRNA and protein determined by northern and western blotting. C. Effect of cycloheximide (CX) and actinomycin D (ActD) on BHA-induced HO-1 expression. SMC were treated with BHA (300 µM) in the presence or absence of CX (5 µg/ml) or ActD (2 µg/ml) for 24 h, and HO-1 mRNA and protein determined by northern and western blotting, respectively. Results were quantified by scanning laser densitometry, expressed in arbitrary units (a.u.), and normalized with respect to housekeeping genes or proteins. Results are means±SEM of 3 experiments. *Statistically significant (p<0.01) increase in HO-1 expression. +Statistically significant (p<0.05) effect of ActD.

 
To further examine the molecular mechanism by which BHA induces HO-1 expression, vascular SMC were transiently transfected with an HO-1 promoter construct and promoter activity monitored. Treatment of SMC with BHA for 24 h stimulated a concentration-dependent increase in HO-1 promoter activity (Fig. 2A). Interestingly, mutation of the ARE (M739) attenuated basal activity and abrogated the response to BHA, suggesting that BHA activates HO-1 transcription via the ARE. Since the transcription factor Nrf2 appears crucial for ARE-mediated gene expression [30], we investigated whether Nrf2 was involved in the activation of HO-1 by BHA. Transfection of vascular SMC with a dominant-negative mutant of Nrf2 that had its activation domain deleted inhibited the BHA-mediated increase in HO-1 promoter activity (Fig. 2A). Moreover, incubation of SMC with BHA stimulated an increase in Nrf2 protein (Fig. 2B). A marked increase in Nrf2 protein was observed after 4 and 8 h of BHA exposure and this increase persisted for 24 h.


Figure 2
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  		Fig. 2 BHA stimulates HO-1 promoter activity and Nrf2 expression in vascular SMC. A. Effect of BHA on HO-1 promoter activity. SMC were co-transfected with a mouse HO-1 promoter construct (E1) or a mutated mouse HO-1 promoter construct (M739) and a β-galactosidase construct (pCMVβgal) to control for transfection efficiency. After 24 h, SMC were exposed to BHA (100 or 300 µM) for 24 h and then analyzed for luciferase and β-galactosidase activity. In some experiments a dominant-negative mutant of Nrf2 (dnNrf2) construct was co-transfected into SMC. Results are means±SEM of 4 separate experiments. *Statistically significant (p<0.01) increase in promoter activity. B. Effect of BHA on Nrf2 protein expression. SMC were treated with BHA (300 µM) for various times (0–24 h) and Nrf2 protein determined by western blotting. Results were quantified by scanning laser densitometry, expressed in arbitrary units (a.u.), and normalized with respect to β-actin. Results are means±SEM of 3 experiments. *Statistically significant (p<0.05) increase in Nrf2 expression.

 
In order to identify the upstream signaling molecules used by BHA to stimulate Nrf2 and HO-1 expression, we analyzed the effect of BHA on various signaling pathways. BHA rapidly stimulated the activation of ERK1/2 and p38 MAPK, as well as phosphatidylinositol-3-kinase (PI3K), as demonstrated by the phosphorylation of its downstream target, Akt (Fig. 3A). Activation of these kinases by BHA peaked between 5 and 10 min. In contrast, BHA failed to stimulate JNK1/2 activation. Interestingly, preincubation of SMC with the p38 MAPK inhibitor SB203580 for 30 min blocked the ability of BHA to activate p38 MAPK in a concentration-dependent manner (Fig. 3B) and prevented the BHA-mediated induction of Nrf2 protein and HO-1 mRNA (Fig. 3C and E). In contrast, the ERK1/2 inhibitor, PD98059, the PI3K/Akt inhibitor LY294002, or the JNK1/2 inhibitor SP600125, had no effect on BHA-induced increases in Nrf2 protein or HO-1 mRNA expression (Fig. 3C, D and E). The effectiveness of all inhibitors for their respective enzymes was confirmed in BHA-treated vascular SMC (data not shown).


Figure 3
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  		Fig. 3 Regulation of BHA-mediated Nrf2 and HO-1 expression by p38 MAPK in vascular SMC. A. Effect of BHA on MAPK and PI3K activity. SMC were treated with BHA (300 µM) for various times (0–30 min) and the activation of ERK1/2, p38 MAPK, JNK1/2, and Akt determined by western blotting using phospho-specific antibodies. Results were quantified by scanning laser densitometry, expressed in arbitrary units (a.u.), and normalized with respect to β-actin. Results are means±SEM of between 3–4 experiments. *Statistically significant (p<0.05) increase in protein activity. B. Effect of SB203580 on BHA-stimulated p38 MAPK activation. SMC were pretreated with SB203580 (SB; 10 or 30 µM) for 30 min and then treated with ({blacksquare}) or without ({square}) BHA (300 µM) for 5 min. Results were quantified by scanning laser densitometry, expressed in a.u., and normalized with respect to total p38 MAPK. Results are means±SEM of 3 experiments. *Statistically significant (p<0.05) increase in p38 MAPK activity. C. Effect of SB203580 and PD98059 on BHA-stimulated Nrf2 protein expression. SMC were pretreated with PD98059 (PD; 30 µM) or SB203580 (SB; 30 µM) for 30 min and then treated with ({blacksquare}) or without ({square}) BHA (300 µM) for 8 h, and Nrf2 protein determined by western blotting. Results were quantified by scanning laser densitometry, expressed in a.u., and normalized with respect to β-actin. Results are means±SEM of 3 experiments. *Statistically significant (p<0.01) increase in Nrf2 protein. D. Effect of LY294002 and SP203580 on BHA-stimulated Nrf2 protein expression. SMC were pretreated with LY294002 (LY; 10 µM) or SP600125 (SP; 20 µM) for 30 min and then treated with ({blacksquare}) or without ({square}) BHA (300 µM) for 8 h, and Nrf2 protein determined by western blotting. Results were quantified by scanning laser densitometry, expressed as a.u., and normalized with respect to β-actin. Results are means±SEM of 4 experiments. *Statistically significant (p<0.01) increase in Nrf2 protein. E. Effect of MAPK and PI3K inhibitors on BHA-stimulated HO-1 mRNA expression. SMC were pretreated with PD (30 µM), SB (30 µM), SP (20 µM) or LY (10 µM) for 30 min and then treated with BHA (300 µM) for 24 h, and HO-1 mRNA determined by northern blotting. Results were quantified by scanning laser densitometry, expressed in a.u., and normalized with respect to GAPDH mRNA. Results are means±SEM of 3 experiments. *Statistically significant (p<0.01) increase in HO-1 mRNA.

 
In subsequent experiments, the functional role of BHA in vascular SMC was examined. Flow cytometry experiments found that BHA blocked the progression of SMC through the cell cycle. Administration of serum for 24 h stimulated cell cycle progression and decreased the population of SMC in the G0/G1 phase of the cell cycle while the number of cells in S and G2M increased (Fig. 4A). However, BHA induced a concentration-dependent increase in the fraction of cells in G0/G1 and this was accompanied by a decrease in the percentage of cells in S and G2M. BHA at a concentration of 300 µM decreased the number of cells in S phase by nearly 80% (Fig. 4B). No apparent toxicity with these doses (≤300 µM) of BHA was noted, as reflected by the lack of a sub-G0/G1 fraction (Fig. 4A). BHA also significantly inhibited serum-stimulated DNA synthesis (Fig. 4C). Interestingly, the HO inhibitor tin protoporphyrin-IX reversed the antiproliferative effect BHA (Fig. 4C). In the absence of serum, BHA or tin protoporphyrin-IX minimally affected DNA synthesis (data not shown).


Figure 4
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  		Fig. 4 BHA inhibits cell cycle progression and DNA synthesis in vascular SMC. A. Representative histograms of SMC DNA content in arbitrary units in cells treated with serum (5%) for 24 h in the presence or absence of BHA (0–300 µM). Data are representative of 3 separate experiments. B. Effect of BHA on the distribution of SMC in the cell cycle. SMC were treated with serum (5%) in the presence or absence of BHA (300 µM). Results are means±SEM of 5 separate experiments. *Statistically significant (p<0.05) effect of BHA. C. Effect of BHA on DNA synthesis. SMC were grown in serum-free (SF) media or treated with a combination of serum (5%), BHA (300 µM), and/or tin protoporphyrin-IX (SnPP; 10 µM). Results are means±SEM of 4 separate experiments. *Statistically significant (p<0.05) increase in [3H]thymidine incorporation. +Statistically significant (p<0.05) effect of BHA.

 
Finally, the effect of BHA on neointimal proliferation was investigated in an in vivo rat model of carotid arterial injury. Fig. 5A illustrates representative cross-sections of perfusion-fixed, Verhoffs-Van-Geisson-stained tissues obtained from sham-injured or injured animals 2 weeks after balloon injury. Morphometric measurements revealed minimal intimal thickness in sham-injured vessels (0.0014±0.0002 mm); however, arterial injury markedly increased neointimal thickness in control injured carotid arteries (0.0635±0.009 mm) and this was significantly reduced in BHA-treated vessels (0.0259±0.008 mm) (Fig. 5B). Furthermore, intimal area was significantly increased from 0.0002±0.00009 mm2 in the sham group to 0.149±0.029 mm2 in the control injured group. Neointimal area was reduced by approximately 50% in the injured group treated with the BHA-containing gel (0.071±0.021 mm2). Similarly, the intima/media area ratio was increased from 0.0015±0.0007 in sham vessels to 0.826±0.116 in the control injured arteries, and this was reduced by nearly 50% in the BHA-treated vessels (0.440±0.129). Interestingly, arterial injury increased the medial area from 0.132±0.006 mm2 to 0.159±0.011 mm2 and this was not changed by BHA (0.159±0.009 mm2). To address possible effects of BHA on the adventitia we performed morphometric measurements of adventitial area and thickness. However, BHA did not affect either adventitial thickness (0.0461±0.0037 mm in the BHA group versus 0.0466±0.0036 mm in the control injured group) or area (0.135±0.013 mm2 in the BHA group versus 0.132±0.011 mm2 in the control injured group). In addition, there was no apparent vascular toxicity associated with the use of BHA, as reflected by tissue necrosis, or infiltration of inflammatory cells, as determined by the lack of staining for the leukocyte marker, CD45. TUNEL staining for apoptosis demonstrated minimal cell death and no difference in the rate of apoptosis between BHA-treated (3.1±0.4%) and control vessels (2.7±0.4%). Finally, perivascular application of BHA stimulated the expression of HO-1 in balloon-injured arteries (Fig. 5C).


Figure 5
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  		Fig. 5 BHA inhibits neointima formation and stimulates HO-1 expression after rat carotid artery injury. A. Representative cross-sections of sham-injured and injured carotid arteries treated with an empty gel (E) or a gel containing BHA (1 mg) 2 weeks after injury (magnification 100x). B. Morphometric measurements of neointima formation in sham-injured or injured carotid arteries 2 weeks after injury. Results are means±SEM of between 7 and 11 carotid arteries. *Statistically significant (p<0.001) effect of arterial injury. +Statistically significant (p<0.05) effect of BHA. C. Western blot of HO-1 protein expression in 1 and 3 day injured vessels in the presence (BHA) and absence (E) of a gel containing BHA (1 mg). Protein expression was quantified by scanning densitometry, expressed in arbitrary units (a.u.), and normalized with respect to β-actin. Results are means±SEM of 3 separate experiments. +Statistically significant (p<0.05) effect of BHA.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study demonstrates that BHA stimulates HO-1 gene expression in vascular SMC by inducing the formation of the Nrf2/ARE complex. The induction of Nrf2 and HO-1 is independent of ERK1/2 or PI3K activation but is dependent on the p38 MAPK pathway. In addition, the current investigation reveals that BHA is a potent inhibitor of SMC growth and neointima formation, and that the induction of HO-1 contributes to the antiproliferative action of BHA. These finding suggest a potentially important therapeutic role for BHA in treating occlusive vascular disease.

Treatment of vascular SMC with BHA results in a concentration- and time-dependent increase in HO-1 protein, mRNA, and promoter activity. The induction of HO-1 transcription by BHA requires the presence of AREs since mutation of the AREs abrogates the stimulation of promoter activity by BHA. Although many transcription factors bind to the ARE, recent work indicates that Nrf2 plays a predominant role in ARE-dependent HO-1 gene expression [30]. Consistent with this hypothesis, we observed that BHA increases the level of Nrf2 in SMC. Moreover, we found that transfection of SMC with a dominant-negative mutant of Nrf2 is able to abolish the activation of HO-1 promoter activity in response to BHA. Interestingly, the peak increase in Nrf2 protein precedes the maximum rise in HO-1 protein expression. This delay in HO-1 expression likely reflects the additional time required for the transcription and translation of the HO-1 gene. Our finding that BHA requires the activation of Nrf2 for the induction of HO-1 expression is consistent with an earlier finding showing that BHA-mediated expression of phase II enzymes is impaired in Nrf2 null mice [31]. In addition, several other phenolic compounds stimulate HO-1 expression via the activation of Nrf2, indicating a critical role for this transcription factor in the induction of HO-1 by these agents [32–35].

Since the MAPK and PI3K pathways have been implicated in the activation of Nrf2 [36], we examined whether these kinases are involved in the activation of Nrf2 by BHA. Although BHA activates the ERK and PI3K pathways, they do not contribute to the activation of Nrf2 since pharmacological inhibition of either enzyme has no effect on the expression of Nrf2. Moreover, inhibition of ERK or PI3K fails to block the induction of HO-1 by BHA. In contrast, inhibition of p38 MAPK activity abolishes the BHA-mediated induction of Nrf2 and HO-1. These findings are consistent with earlier studies demonstrating the requirement of p38 MAPK in the activation of Nrf2 by other phenolic antioxidants [33,34] but they contrast with a recent report linking the activation of ERK and JNK signaling pathways with the mobilization of Nrf2 by BHA in hepatic tumor cells [37]. The cause for these divergent results is not known but may reflect differences in cell type and culture conditions.

Although BHA inhibits the proliferation of tumor cells and fibroblasts [18–20], its action on vascular SMC growth has not been determined. In the current study, we found that BHA is a potent inhibitor of cell cycle progression. In particular, BHA arrests SMC in the G0/G1 phase of the cell cycle preventing their entry into S phase. Consistent with the cell cycle distribution experiments, BHA blocks serum-stimulated DNA synthesis. The concentration of BHA (100 and 300 µM) required to block SMC mitogenesis is similar to what has been reported in other cell types, and is below the cytotoxic dose [19,20,38]. In addition, the ability of BHA to suppress DNA synthesis is substantially reversed by the HO inhibitor, tin protoporphyrin-IX, indicating that the induction of HO-1 contributes to the antiproliferative action of BHA. The ability of HO-1 to block SMC growth is likely mediated via the formation of carbon monoxide and/or the bile pigments biliverdin and bilirubin, which have been demonstrated to halt SMC in the G0/G1 phase of the cell cycle [7,39,40]. Interestingly, HO-1 also underlies the growth suppressive property of the phenolic compound probucol, suggesting that the induction of HO-1 represents a central mechanism by which phenolic derivatives block SMC growth [41].

We further explored the effect of BHA on vascular SMC proliferation using the well-established rat carotid artery injury model. This model is characterized by its high degree of reproducibility and with the development of SMC-rich intimal lesions. BHA was applied topically to the adventitia of the blood vessel via a specific local delivery copolymer that we and others have successfully used to deliver drugs to the vessel wall [11,26–28]. This local delivery system allows for the continuous discharge of BHA over the course of several days and avoids possible non-specific actions associated with the systemic administration of the drug. We found that local administration of BHA significantly blocks intimal thickening following balloon injury and this is associated with a prominent increase in HO-1 protein expression in the vessel wall. Significantly, the blockade of neointima formation by BHA occurs in the absence of any overt sign of cell necrosis or apoptosis, suggesting that vessel wall BHA concentrations were below the cytotoxic range. Thus, the local application of BHA represents an attractive therapeutic agent for attenuating the vascular response to injury and may prove highly effective in preventing restenosis when liberated from the surface of a stent.

Our finding that BHA ameliorates vascular remodeling following balloon injury is consistent with recent reports demonstrating the beneficial effect of phenolic compounds in pathologic remodeling states. In particular, BHA inhibits the development of hyperplastic lesions in an animal model of pulmonary fibrosis [21]. Furthermore, the phenolic antioxidant probucol protects against the development of atherosclerotic lesions in apoE-deficient mice and markedly attenuates the formation of a neointima following arterial injury of rabbits and obese Zucker rats [41,42]. In all instances, the decrease in lesion formation by probucol is dependent on the induction of HO-1. Similarly, polyphenolic compounds such as curcumin and resveratrol, which are established inducers of HO-1 [33,34], block neointima formation after arterial injury in various animal models [43,44]. Interestingly, phenolic antioxidants which fail to stimulate HO-1 expression do not confer any protection against arterial lesion formation, underscoring the crucial role of HO-1 in mediating the atheroprotective effects of these compounds [42]. More generally, it is tempting to speculate that the cardioprotection associated with the consumption of phenol-rich foods may, in part, be attributed to their ability to induce HO-1 [45].

In conclusion, the present study demonstrates that BHA induces HO-1 gene expression via the p38 MAPK-mediated activation of Nrf2. In addition, it was found that the induction of HO-1 contributes to the ability of BHA to inhibit SMC growth and neointima formation following arterial balloon injury in rats. These findings further reinforce the notion that HO-1 is a critical regulator of vascular remodeling, and highlight the therapeutic potential for using specific phenolic antioxidants in the treatment of occlusive vascular disease.


   Acknowledgements
 
We gratefully acknowledge Charlene Thomson for the help with the flow cytometry experiments. This work was supported by the National Institutes of Health grants HL59976, HL74966, and HL62467.


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Schwartz S.M., Liaw L. Growth control and morphogenesis in the development and pathology of arteries. J Cardiovasc Pharmacol (1993) 21(Suppl. 1):S31–S49.[Web of Science][Medline]
  2. Dzau V.J., Braun-Dellaeus R.C., Shedding D.G. Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies. Nat Med (2002) 8:1249–1256.[CrossRef][Web of Science][Medline]
  3. Holmes D.R. Jr. State of the art in coronary interventions. Am J Cardiol (2003) 135:955–959.
  4. Maines M.D. Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications. FASEB J (1989) 2:2557–2568.[Web of Science]
  5. Duckers H.J., Boehm M., True A.L., Yet S.-F., Park J.L., Webb R.C., et al. Heme oxygenase-1 protects against vascular constriction and proliferation. Nat Med (2001) 7:693–698.[CrossRef][Web of Science][Medline]
  6. Morita T., Kourembanas S. Endothelial cell expression of vasoconstrictors and growth factors is regulated by smooth muscle cell-derived carbon monoxide. J Clin Invest (1995) 96:2676–2682.[Web of Science][Medline]
  7. Peyton K.J., Reyna S.V., Chapman G.B., Ensenat D., Liu X., Wang H., et al. Heme oxygenase-1-derived carbon monoxide is an autocrine inhibitor of vascular smooth muscle cell growth. Blood (2002) 99:4443–4448.[Abstract/Free Full Text]
  8. Togane Y., Toshisuki M., Suematsu M., Ishimura Y., Yamazaki J., Katayama S. Protective roles of endogenous carbon monoxide in neointimal development elicited by arterial injury. Am J Physiol Heart Circ Physiol (2000) 278:H623–H632.[Abstract/Free Full Text]
  9. Aizawa T., Ishizaka N., Taguchi J., Kimura S., Kurokawa K., Ohno M. Balloon injury does not induce heme oxygenase-1 gene expression, but administration of hemin inhibits neointimal formation in balloon injured rat carotid arteries. Biochem Biophys Res Commun (1999) 261:302–307.[CrossRef][Web of Science][Medline]
  10. Tulis D.A., Durante W., Peyton K.J., Evans A.J., Schafer A.I. Heme oxygenase-1 attenuates vascular remodeling following balloon injury in rat carotid arteries. Atherosclerosis (2001) 155:113–122.[CrossRef][Web of Science][Medline]
  11. Tulis D.A., Durante W., Liu X., Evan A.J., Peyton K.J., Schafer A.I. Adenovirus-mediated heme oxygenase-1 gene delivery inhibits injury-induced vascular neointima formation. Circulation (2001) 104:2710–2715.[Abstract/Free Full Text]
  12. Yet S.F., Layne M.D., Liu X., Chen Y.H., Ith B., Sibinga N.E., et al. Absence of heme oxygenase-1 exacerbates atherosclerosis lesion formation an vascular remodeling. FASEB J (2003) 17:1759–1761.[Abstract/Free Full Text]
  13. Exner M., Schillinger M., Minar E., Mlekusch W., Schlerka G., Haumer M., et al. Heme oxygenase-1 gene promoter microsatellite polymorphism is associated with restenosis after percutaneous transluminal angioplasty. J Endovasc Ther (2001) 8:433–440.[CrossRef][Web of Science][Medline]
  14. Chen Y.-H., Chau L.-Y., Lin M.-W., Chen L.-C., Yo M.-H., Chen J.-W., et al. Heme oxygenase-1 gene promoter microsatellite polymorphism is associated with angiographic restenosis after coronary stenting. Eur Heart J (2004) 25:39–47.[Abstract/Free Full Text]
  15. Kahl R. Synthetic antioxidants: biochemical action and interference with radiation, toxic compounds, chemical mutagens and chemical carcinogens. Toxicology (1984) 59:179–194.[CrossRef]
  16. Benson A.M., Hunkeler M.J., Talalay P. Increase of NAD(P)H :quinine reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity. Proc Natl Acad Sci U S A (1980) 77:5216–5220.[Abstract/Free Full Text]
  17. De Long M.J., Prochaska H.J., Talalay P. Tissue-specific induction patterns of cancer-protective enzymes in mice by tert-butyl-4-hydroxyanisole and related substituted phenols. Cancer Res (1985) 45:546–551.[Abstract/Free Full Text]
  18. Saito M., Sakagami H., Fujisawa S. Cytotoxicity and apoptosis induction by butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Anticancer Res (2003) 23:4693–4701.[Web of Science][Medline]
  19. Taniguchi S., Furukawa M., Kono T., Hisa T., Ishii M., Hamada T. Butylated hydroxyanisole blocks the inhibitory effects of tumor necrosis factor-{alpha} on collagen production in human dermal fibroblasts. J Dermatol Sci (1996) 12:44–49.[CrossRef][Web of Science][Medline]
  20. Brekke O.L., Shalaby M.R., Sundan A., Espevik T., Bjerve K.S. Butylated hydroxyanisole specifically inhibits tumor-necrosis factor-induced cytotoxicity and growth enhancement. Cytokine (1992) 4:269–280.[CrossRef][Web of Science][Medline]
  21. Ikezaki S., Nishikawa A., Enami T., Furukawa F., Imazawa T., Uneyama C., et al. Inhibitory effects of the dietary antioxidants butylated hydroxyanisole and butylated hydroxytoluene on bronchioloalveolar cell proliferation during the bleomycin-induced pulmonary fibrosing process in hamsters. Food Chem Toxicol (1996) 34:327–335.[CrossRef][Web of Science][Medline]
  22. Durante W., Schini V.B., Catovsky S., Kroll M.H., Vanhoutte P.M., Schafer A.I. Plasmin potentiates the induction of nitric oxide synthase by interleukin-1β in vascular smooth muscle. Am J Physiol Heart Circ Physiol (1993) 264:H617–H623.[Abstract/Free Full Text]
  23. Durante W., Kroll M.H., Christodoulides N., Peyton K.J., Schafer A.I. Nitric oxide induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells. Circ Res (1997) 80:557–564.[Abstract/Free Full Text]
  24. Alam J., Wicks C., Stewart D., Gong P., Touchard C., Otterbein S., et al. Mechanism of heme oxygenase-1 gene activation by cadmium in MCF-7 mammary epithelial cells: role of p38 kinase and Nrf2 transcription factor. J Biol Chem (2000) 275:27694–27702.[Abstract/Free Full Text]
  25. Liu X., Chapman G.B., Peyton K.J., Schafer A.I., Durante W. Carbon monoxide inhibits apoptosis in vascular smooth muscle cells. Cardiovasc Res (2002) 55:396–405.[Abstract/Free Full Text]
  26. Granada J.F., Ensenat D., Keswani A.N., Kaluza G.L., Raizner A.E., Liu X.M., et al. Single perivascular delivery of mitomycin C stimulates p21 expression and inhibits neointima formation in rat arteries. Arterioscler Thromb Vasc Biol (2005) 25:2343–2348.[Abstract/Free Full Text]
  27. Fulton G.J., Davies M.G., Barber L., Svendsen E., Hagen P.O. Locally applied antisense oligonucleotide to proliferating cell nuclear antigen inhibits intimal thickening in experimental vein grafts. Ann Vasc Surg (1998) 12:412–417.[CrossRef][Web of Science][Medline]
  28. Hu Y., Zou Y., Dietrich H., Wick G., Xu Q. Inhibition of neointima hyperplasia in mouse vein grafts by locally applied suramin. Circulation (1999) 100:861–868.[Abstract/Free Full Text]
  29. Abe T., Sasaki M., Nakajima H., Ogita M., Naitou H., Nagase A., et al. Evaluation of pluronic F127 as a gradual release of anticancer drug. Gan To Kagaku Ryoho (1990) 17:1546–1550.[Medline]
  30. Alam J., Cook J.L. Transcriptional regulation of the heme oxygenase-1 gene via the stress response pathway. Curr Pharm Des (2003) 9:2499–2511.[CrossRef][Web of Science][Medline]
  31. Noda S., Harada N., Hida A., Fujii-Kuriyama Y., Motohashi, Yamamoto M. Gene expression of detoxifying enzymes in AhR and Nrf2 compound null mutant mice. Biochem Biophys Res Commun (2003) 303:105–111.[CrossRef][Web of Science][Medline]
  32. Alam J., Stewart D., Touchard C., Boinapally S., Choi A.M.K., Cook J.L. Nrf2, a cap'n'collar transcription factor, regulates induction of the heme oxygenase-1 gene. J Biol Chem (1999) 274:26071–26078.[Abstract/Free Full Text]
  33. Balogun E., Hoque M., Gong P., Killeen E., Green C.J., Foresti R., et al. Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element. Biochem J (2003) 371:887–895.[CrossRef][Web of Science][Medline]
  34. Martin D., Rojo A.I., Salinas M., Diaz R., Gallardo G., Alam J., et al. Regulation of heme oxygenase-1 expression through the phosphatidlyinositol-3-kinase/Akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnasol. J Biol Chem (2004) 279:8919–8929.[Abstract/Free Full Text]
  35. Chen C.Y., Jang J.H., Li M.H., Surh Y.J. Resveratrol upregulates heme oxygenase-1 expression via activation of NF-E2-related factor 2 in PC12 cells. Biochem Biophys Res Commun (2005) 331:993–1000.[CrossRef][Web of Science][Medline]
  36. Nguyen T., Yang C.S., Pickett C.B. The pathways and molecular mechanisms regulating Nrf2 activation in response to chemical stress. Free Radic Biol Med (2004) 37:433–441.[CrossRef][Web of Science][Medline]
  37. Yuan X., Xu C., Pan Z., Keum Y.-S., Kim J.-H., Shen G., et al. Butylated hydroxyanisole regulates ARE-mediated gene expression via Nrf2 coupled with ERK and JNK signaling pathway in HepG2 cells. Mol Carcinog (2006) 45:841–850.[CrossRef][Web of Science][Medline]
  38. Yu R., Manlekar S., Tony Kong A.-N. Molecular mechanisms of butylated hydroxyanisole-induced toxicity: induction of apoptosis through direct release of cytochrome c. Mol Pharmacol (2000) 58:431–437.[Abstract/Free Full Text]
  39. Otterbein L.E., Zuckerbraun B.S., Haga M., Liu F., Song R., Usheva A., et al. Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury. Nat Med (2003) 9:183–190.[CrossRef][Web of Science][Medline]
  40. Ollinger R., Bilban M., Erat A., Froio A., McDaid J., Tyagi S., et al. Bilirubin: a natural inhibitor of vascular smooth muscle cell proliferation. Circulation (2005) 112:1030–1039.[Abstract/Free Full Text]
  41. Yeng Y.-M., Wu B.J., Witting P.K., Stocker R.S. Probucol protects against smooth muscle cell proliferation by upregulating heme oxygenase-1. Circulation (2004) 110:1855–1860.[Abstract/Free Full Text]
  42. Wu B.J., Kathir K., Witting P.K., Beck K., Choy K., Li C., et al. Antioxidants protect from atherosclerosis by a heme oxygenase-1 pathway that is independent of free radical scavenging. J Exp Med (2006) 203:1117–1127.[Abstract/Free Full Text]
  43. Yang X., Thomas D.P., Zhang X., Culver B.W., Alexander B.M., Murdoch W.J., et al. Curcumin inhibits platelet-derived growth factor-stimulated vascular smooth muscle cell function and injury-induced neointima formation. Arterioscler Thromb Vasc Biol (2006) 26:85–90.[Abstract/Free Full Text]
  44. Zou J., Huang Y., Cao K., Yang G., Yin H., Len J., et al. Effect of resveratrol on intimal hyperplasia after endothelium denudation in an experimental rabbit model. Life Sci (2000) 68:153–163.[CrossRef][Web of Science][Medline]
  45. Zern T.L., Fernandez M.L. Cardioprotective effects of dietary polyphenols. J Nutr (2005) 135:2291–2294.[Abstract/Free Full Text]

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