Cardiovascular Research

Cardiovascular Research 2002 55(2):396-405; doi:10.1016/S0008-6363(02)00410-8
© 2002 by European Society of Cardiology

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Copyright © 2002, European Society of Cardiology

Carbon monoxide inhibits apoptosis in vascular smooth muscle cells

Xiao-ming Liu^b, Gary B Chapman^b, Kelly J Peyton^a, Andrew I Schafer^a,b and William Durante^a,b,c,*

^aHouston VA Medical Center, Building 109, Room 130, 2002 Holcombe Blvd, Houston, TX 77030, USA
^bDepartment of Medicine, Baylor College of Medicine, Houston, TX 77030, USA
^cDepartment of Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA

^* Corresponding author. Tel.: +1-713-791-1414x5824; fax: +1-713-794-7165 wdurante{at}bcm.tmc.edu

Received 27 November 2001; accepted 26 March 2002

	Abstract

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

 
Objective: Carbon monoxide (CO) is generated from vascular smooth muscle cells via the degradation of heme by the enzyme heme oxygenase-1. Since smooth muscle cell apoptosis is associated with numerous vascular disorders, we investigated whether CO regulates apoptosis in vascular smooth muscle. Methods and Results: Treatment of cultured rat aortic smooth muscle cells with a combination of cytokines (interleukin-1β, 5 ng/ml; tumor necrosis factor-, 20 ng/ml; interferon-, 200 U/ml) for 48 h stimulated apoptosis, as demonstrated by DNA laddering, annexin V binding, and caspase-3 activation. However, the exogenous administration of CO inhibited cytokine-mediated apoptosis. The antiapoptotic action of CO was partially dependent on the activation of soluble guanylate cyclase and was associated with the inhibition of mitochondrial cytochrome c release and with the suppression of p53 expression. Incubation of smooth muscle cells with the cytokines also resulted in a pronounced increase in heme oxygenase-1 protein after 24 h of stimulation. The addition of the heme oxygenase inhibitor, zinc protoporphyrin-IX, or the CO scavenger, hemoglobin, stimulated apoptosis following 24 h of cytokine exposure. Conclusions: These results demonstrate that CO, either administered exogenously or endogenously derived from heme oxygenase-1 activity, inhibits vascular smooth muscle cell apoptosis. The ability of CO to block smooth muscle cell apoptosis may play an important role in blocking lesion formation at sites of vascular injury.

KEYWORDS Apoptosis; Arteries; Cell culture/isolation; Cytokines; Smooth muscle

	1. Introduction

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

 
Carbon monoxide (CO) is a diatomic gas that is generated primarily from the degradation of heme by the enzyme heme oxygenase (HO) [1]. Three distinct isoforms of HO have been identified [2,3]. These isozymes are products of different genes and differ markedly in their tissue distribution as well as their molecular properties. The HO-2 isoform is constitutively expressed and is present in high levels in the brain and testes [2]. The recently discovered HO-3 isoform is closely related to HO-2 but is nearly devoid of catalytic activity and may function as a heme-sensing or heme-binding protein [3]. In contrast, the HO-1 isozyme is ubiquitously distributed and is highly inducible [2]. HO-1 has been considered to play an important cytoprotective role in defense against cellular stress [4].

Recent studies indicate that HO-1-derived CO has significant physiological mediator functions in the circulation. The exogenous administration of CO relaxes blood vessels isolated from various vascular sources and animal species [5]. Moreover, induction of HO-1 causes a marked decrease in blood pressure in hypertensive rats whereas HO-1 inhibition increases blood pressure, suggesting that endogenous CO subserves a tonic vasodepressor function [6,7]. HO-1-derived CO also blocks the synthesis of growth factors from vascular cells and directly inhibits vascular smooth muscle cell (SMC) proliferation [8,9]. In addition, the HO-1-catalyzed release of CO from vascular cells blocks platelet aggregation, indicating a potentially important antithrombotic role for this gas [10].

Apoptosis, or programmed cell death, plays a pivotal role in both normal development and pathobiology of the vascular system. Vascular SMC apoptosis is a prominent feature of the remodeling process that occurs in atherosclerosis, hypertension, and restenosis following angioplasty [11–13]. Apoptosis is associated with unique morphological changes, including DNA fragmentation, condensation of cytoplasm, redistribution of membrane phospholipids, cell budding, and activation of caspases [14]. Since SMC apoptosis may contribute to the remodeling response following vascular injury, identification of both the negative and positive modulators of apoptosis may lead to novel therapeutic approaches in treating vascular disease. In the present study, we examined whether CO regulates apoptosis in vascular SMC. We now report that CO, either exogenously administered or endogenously generated by HO-1 activity, is a potent inhibitor of vascular SMC apoptosis. The antiapoptotic effect of CO is dependent, in part, on the activation of soluble guanylate cyclase and is associated with inhibition of the expression of the tumor suppressor protein p53 and inhibition of the release of cytochrome c from the mitochondria. The ability of CO to block SMC apoptosis may function to promote tissue repair and limit lesion formation at sites of vascular injury.

	2. Methods

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

 
2.1 Materials
Minimum essential media, serum, elastase, collagenase, penicillin, streptomycin, Tris, Tes, HEPES, SDS, β-mercaptoethanol, phenol, chloroform, EDTA, Triton X-100, agarose, sucrose, desferrioxamine, iron, trypan blue, methyl-L-arginine (L-NMA), hemoglobin, and sodium acetate were from Sigma Chemical Company (St. Louis, MO); RNase A, proteinase K, and phenylmethylsulfonyl fluoride (PMSF) were from Boehringer Mannheim (Indianapolis, IN); recombinant mouse tumor necrosis factor- and interferon- were purchased from Genzyme (Cambridge, MA); recombinant mouse interleukin-1β was purchased from R&D Systems (Minneapolis, MN); zinc protoporphyrin-IX (ZnPP-IX) was from Porphyrin Products (Logan, UT); 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was from Calbiochem-Novabiochem Corporation (La Jolla, CA); a polyclonal HO-1 antibody was from StressGen (Victoria, Canada); a monoclonal p53 antibody was from Pharmingen (San Diego, CA); polyclonal antibodies against p38 mitogen-activated protein kinase (MAPK) and phosphorylated p38 MAPK were from Cell Signaling Technology (Beverly, MA).

2.2 Cell culture
Vascular SMC were isolated by elastase and collagenase digestion of thoracic aortae obtained from 12-week-old male rats and characterized by immunological and morphological criteria [15]. 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). Cells were serially cultured in minimum essential medium containing 10% serum, Earle's salts, 5.6 mM glucose, 2 mM L-glutamine, 20 mM Tes, 20 mM Hepes, and 100 units/ml of penicillin, streptomycin, and neomycin. Subcultured cells were used between passages 5 and 22.

2.3 CO exposure
SMC were exposed to CO via a previously described environmental chamber [16]. CO at a concentration of 1% in air was mixed with air containing 5% CO₂ in a stainless steel mixing cylinder prior to delivery into the humidified 37 °C environmental chamber. Flow into the chamber was at 1 l/min and CO levels were continuously monitored by electrochemical detection using a CO analyzer (Interscan, Chatsworth, CA).

2.4 DNA laddering
SMC were incubated in Tris buffer (50 mM Tris (pH 7.4), 10 mM EDTA, 0.5% Triton X-100) for 15 min on ice. The lysate was centrifuged at 13,000xg for 30 min at 4 °C, supernatant collected, and incubated with RNase A (50 µg/ml) for 1 h at 37 °C. Proteinase K (100 µg/ml) and SDS (0.5%) were then added and incubated for 3 h at 50 °C. DNA was extracted with phenol (pH 8.0) followed by phenol:chloroform (1:1, v/v) and chloroform. The aqueous phase was precipitated with 2.0 volumes of ice-cold ethanol and 0.1 volume of 3 M sodium acetate (pH 5.2) at –80 °C, overnight. The precipitates were collected by centrifugation at 13,000xg for 30 min at 4 °C, washed with 70% ethanol, and air-dried. The pellets were resuspended in Tris (10 mM, pH 8.0)/EDTA (1 mM) buffer and loaded onto 2% agarose gels containing ethidium bromide. Electrophoresis was performed for 30 min at 100 V and photographed under UV illumination.

2.5 Annexin V binding
The movement of phosphatidylserine to the extracellular surface was determined by annexin V binding using a commercially available kit (R&D Systems, Minneapolis, MN) [17]. Briefly, SMC were harvested with trypsin (0.025%)/EDTA (1 mM) and suspended in binding buffer at a final cell concentration of 10⁶ cells/ml. Approximately 10⁵ cells were transferred to a flow cytometric vial, and propidium iodide and fluorescein-conjugated annexin V were added to the suspension. The suspension was vortexed and incubated in the dark for 15 min and then analyzed in a Becton-Dickinson FACScan flow cytometer (Bedford, MA).

2.6 Caspase-3 activity
Caspase-3 activity was measured using a colorimetric assay by Clontech (Palo Alto, CA) [17], which monitors the cleavage of the p-nitroanilide-conjugated caspase-3 substrate, DEVD. SMC (2x10⁶ cells) were trypsinized, washed in ice-cold PBS, and suspended in lysis buffer (50 mM Hepes (pH 7.5), 10% sucrose, 0.1% Triton X-100) on ice for 10 min. After centrifugation at 13,000xg for 5 min at 4 °C, supernatants were incubated with 50 µM of DVED and absorbance measured at 405 nm using a µQuant spectrophotometer (Bio-Tek Instruments, Winooski, VT).

2.7 Cell viability
Cell viability was monitored by measuring the uptake of the membrane-impermeable stains propidium iodide and trypan blue, as we have previously described [15,17].

2.8 Western blotting
SMC were lysed in electrophoresis buffer (125 mM Tris (pH 6.8), 12.5% glycerol, 2% SDS, and 1 mM dithiothreitol), boiled and sonicated. The lysate was centrifuged at 14,000xg for 15 min at 4 °C, the supernatant collected and SDS–PAGE was performed. The separated blots were electrophoretically transferred to nitrocellulose membranes and blocked overnight at 4 °C in PBS containing Tween 20 (0.1%) and nonfat milk (5%). Blots were then incubated with either the p53 (10 µg/ml), HO-1 (1:500), p38 MAPK (1:500), or phospho-specific p38 MAPK (1:500) antibody for 1 h. Membranes were then washed in PBS and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit (1:7500) or goat anti-mouse (1:5000 dilution) antibody. After further washing with PBS, blots were developed using the ECL method (Amersham, Arlington Heights, IL). Relative protein levels were quantified by scanning densitometry (LKB Ultrascan XL laser densitometer, Bromma, Sweden).

2.9 Mitochondrial cytochrome c release
SMC were washed in ice-cold PBS and resuspended in Hepes buffer (20 mM Hepes (pH 7.5), 10 mM KCl, 1.5 MgCl₂, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM PMSF) containing 250 mM sucrose on ice for 15 min. The cells were sonicated on ice and the lysates were centrifuged at 750xg for 15 min at 4 °C. The supernatants were then collected and centrifuged at 14,000xg for 15 min at 4 °C and the resulting pellets containing mitochondria (mitochondrial fraction) were resuspended in Hepes buffer containing sucrose (250 mM) and stored at –70 °C. The supernatants were centrifuged at 100,000xg for 1 h at 4 °C and the resulting supernatants (cytosolic fraction) were also stored at –70 °C. Cytochrome c was analyzed by Western blotting, as described above, using a monoclonal antibody (5 µg/ml) (R&D Systems, Minneapolis, MN) that specifically recognizes the denatured form of cytochrome c.

2.10 Statistics
Results are expressed as the mean±S.E.M. Statistical differences between multiple groups were evaluated by an analysis of variance with post hoc Bonferroni's t-test. Values of P<0.05 were considered to be statistically significant.

	3. Results

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

 
Treatment of vascular SMC with a cytokine mixture consisting of interleukin-1β (5 ng/ml), tumor necrosis factor- (20 ng/ml), and interferon- (200 U/ml) induced apoptosis in a time-dependent manner. Although DNA fragmentation was absent in control SMC and in SMC exposed to the cytokine mixture for 24 h, pronounced DNA laddering was observed following 48 h of treatment (Fig. 1A). In addition, the cytokine mixture markedly increased SMC annexin V binding following 48 h of exposure (Fig. 1B). However, exposure of vascular SMC to CO (200 ppm) inhibited cytokine-mediated DNA laddering and annexin V binding (Fig. 2A and B). Incubation of vascular SMC with the cytokine mixture for 48 h also resulted in a significant increase in caspase-3 activity that was reversed by the administration of CO (50–200 ppm) in a concentration-dependent manner (Fig. 2C). CO (50–200 ppm) had no adverse effect on the viability of SMC (data not shown). Finally, treatment of vascular SMC with the bile pigments, biliverdin (5 µM) or bilirubin (5 µM), or free iron (5 µM), had no effect on cytokine-mediated DNA laddering or annexin V binding (Fig. 3).

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Inflammatory cytokines stimulate apoptosis in vascular SMC. (A) DNA laddering in SMC treated with a cytokine mixture (CM) consisting of interleukin-1β (5 ng/ml), tumor necrosis factor- (20 ng/ml), and interferon- (200 U/ml) for 24 or 48 h. Data are representative of four separate experiments. (B) Annexin V binding in SMC treated with a CM for 24 and 48 h. Results are means±S.E.M. of four separate experiments. *Statistically significant effect of CM.

 

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 CO inhibits cytokine-stimulated vascular SMC apoptosis. (A) DNA laddering in SMC treated with a cytokine mixture (CM) consisting of interleukin-1β (5 ng/ml), tumor necrosis factor- (20 ng/ml), and interferon- (200 U/ml) for 48 h in the presence and absence of CO (200 ppm). Data are representative of three separate experiments. (B) Annexin V binding in SMC treated with a CM for 48 h in the presence or absence of CO (200 ppm). Results are means±S.E.M. of three separate experiments. *Statistically significant effect of CM. (C) Caspase-3 activity in SMC treated with a CM for 48 h in the presence and absence of CO (50–200 ppm). Results are means±S.E.M. of four separate experiments. *Statistically significant effect of CM.

 

View larger version (33K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of bile pigments and iron on cytokine-stimulated vascular SMC apoptosis. (A) DNA laddering in SMC treated with a cytokine mixture (CM) consisting of interleukin-1β (5 ng/ml), tumor necrosis factor- (20 ng/ml), and interferon- (200 U/ml) for 48 h in the presence and absence of biliverdin (5 µM), bilirubin (5 µM), or iron (5 µM). Data are representative of four separate experiments. (B) Annexin V binding in SMC treated with a CM for 48 h in the presence or absence of biliverdin (5 µM), bilirubin (5 µM), or iron (5 µM). Results are means±S.E.M. of four separate experiments. *Statistically significant effect of CM.

 
In the next series of experiments, the mechanism by which CO inhibits SMC apoptosis was examined. Treatment of vascular SMC with the soluble guanylate cyclase inhibitor, ODQ (10 µM) [18] reversed the antiapoptotic effect of CO. The CO-mediated inhibition of DNA laddering and annexin V labeling was markedly reduced, but not completely reversed, in the presence of ODQ (Fig. 4A and B). In the absence of CO, ODQ had no effect on DNA laddering or annexin V binding (data not shown). Since a recent study [19] demonstrated that p38 MAPK mediates the antiapoptotic action of CO in endothelial cells we also investigated the involvement of this kinase. Control untreated SMC did not express p38 MAPK activity, however, treatment of SMC with the cytokine mixture resulted in the rapid activation of p38 MAPK that peaked after 15 min (Fig. 5A and B). CO failed to stimulate p38 MAPK activation (data not shown) and did not potentiate the cytokine-mediated activation of p38 MAPK (Fig. 5A and B). Moreover, incubation of SMC with the p38 MAPK inhibitor, SB203580 [20], did not reverse the inhibition of annexin V binding by CO in cytokine-treated SMC (Fig. 5C).

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 ODQ reverses the antiapoptotic effect of CO on vascular SMC. (A) DNA laddering in SMC treated with a cytokine mixture (CM) consisting of interleukin-1β (5 ng/ml), tumor necrosis factor- (20 ng/ml), and interferon- (200 U/ml) for 48 h in the presence and absence of CO (200 ppm) or ODQ (10 µM). Data are representative of five separate experiments. (B) Annexin V binding in SMC treated with a CM for 48 h in the presence and absence of CO (200 ppm) or ODQ (10 µM). Results are means±S.E.M. of five separate experiments. *Statistically significant effect of CM. +Statistically significant effect of ODQ.

 

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Role of p38 MAPK activity on vascular SMC apoptosis. (A) SMC were treated with a cytokine mixture (CM) consisting of interleukin-1β (5 ng/ml), tumor necrosis factor- (20 ng/ml), and interferon- (200 U/ml) for various times (0–30 min) in the presence and absence of CO (200 ppm). Activated p38 MAPK and total p38 MAPK were determined by Western blotting using antibodies that recognize only the phosphorylated or both the phosphorylated and non-phosphorylated forms of p38 MAPK, respectively. Data are representative of four separate experiments. (B) Quantification of p38 MAPK phosphorylation following normalization for total p38 MAPK by laser densitometry after treatment of SMC with a CM for various times (0–30 min) in the presence and absence of CO (200 ppm). Results are means±S.E.M. of four separate experiments. *Statistically significant effect of CM. (C) Annexin V binding in SMC treated with a CM and CO (200 ppm) for 48 h in the presence or absence of SB203580 (10 µM). Results are means±S.E.M. of four separate experiments. *Statistically significant effect of CM.

 
Incubation of vascular SMC with the cytokine mixture stimulated the mitochondrial release of cytochrome c. Western blot analysis revealed that cytochrome c was found exclusively in the membrane fraction in control, untreated SMC (Fig. 6). However, following 48 h of exposure to the cytokine mixture, cytochrome c was also detected in the cytosolic fraction (Fig. 6). The cytokine-mediated translocation of cytochrome c from the membrane to the cytosolic fraction following cytokine treatment was completely blocked by CO (200 ppm) (Fig. 6). Treatment of SMC with the cytokine mixture also increased the expression of p53 protein by approximately twofold (Fig. 6). However, CO (200 ppm) blocked the induction of p53 expression by the inflammatory cytokines (Fig. 6). In the absence of cytokines, CO had no effect on the level of p53 protein (Fig. 6).

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Effect of CO on the cytokine-stimulated release of mitochondrial cytochrome c and p53 expression in vascular SMC. (A) SMC were treated with a cytokine mixture (CM) consisting of interleukin-1β (5 ng/ml), tumor necrosis factor- (20 ng/ml), and interferon- (200 U/ml) for 48 h in the presence and absence of CO (200 ppm). The presence of cytochrome c in the mitochondrial and cytosolic fractions was detected by Western blotting. Data are representative of three separate experiments. (B) Western blot of p53 in SMC treated with the CM for 48 h in the presence and absence of CO (200 ppm). Data are representative of three separate experiments. (C) Quantification of p53 expression by laser densitometry following treatment of SMC with CM for 48 h in the presence and absence of CO (200 ppm). Results are means±S.E.M. of three separate experiments. *Statistically significant effect of CM.

 
Finally, we wished to determine if endogenously generated CO, produced by the activation of HO-1, likewise exerted an antiapoptotic effect. Incubation of SMC with the cytokine mixture resulted in a time-dependent increase in HO-1 protein that peaked following 24 h of exposure (Fig. 7). Densitometric analysis revealed that cytokines stimulated HO-1 protein expression by 23- and 13-fold after 24 and 48 h, respectively. Annexin V binding is not increased after only 24 h of cytokine treatment of SMC. However, the addition of either the HO inhibitor, ZnPP-IX (50 µM) [2], or the CO scavenger, Hb (50 µM), stimulated annexin V binding under these conditions (Fig. 8). In contrast, the addition of the nitric oxide synthase inhibitor, L-NMA (1 mM), blocked the induction of DNA laddering and annexin V binding by the inflammatory cytokines (Fig. 9).

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Cytokine-stimulated HO-1 protein expression in vascular SMC. (A) Western blot of HO-1 in SMC treated with a cytokine mixture (CM) consisting of interleukin-1β (5 ng/ml), tumor necrosis factor- (20 ng/ml), and interferon- (200 U/ml) for the indicated times. Data are representative of three separate experiments. (B) Quantification of HO-1 expression by laser densitometry. Results are means±S.E.M. of three separate experiments. *Statistically significant effect of CM.

 

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Effect of HO-1 activity on vascular SMC apoptosis. Annexin V binding was measured in SMC that were treated with a cytokine mixture (CM) consisting of interleukin-1β (5 ng/ml), tumor necrosis factor- (20 ng/ml), and interferon- (200 U/ml) for 24 h in the presence and absence of ZnPP-IX (50 µM) or hemoglobin (Hb; 50 µM). Results are means±S.E.M. of five separate experiments. *Statistically significant effect of ZnPP-IX or Hb.

 

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 9 L-NMA inhibits cytokine-stimulated vascular SMC apoptosis. (A) DNA laddering in SMC treated with a cytokine mixture (CM) consisting of interleukin-1β (5 ng/ml), tumor necrosis factor- (20 ng/ml), and interferon- (200 U/ml) for 48 h in the presence and absence of L-NMA (1 mM). Data are representative of four separate experiments. (B) Annexin V binding in SMC treated with a CM for 48 h in the presence or absence of L-NMA (1 mM). Results are means±S.E.M. of four separate experiments. *Statistically significant effect of CM. +Statistically significant effect of L-NMA.

 

	4. Discussion

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

 
Apoptosis is a highly regulated process of cell death that may contribute to the development of vascular disease. In the current study, we demonstrate that CO is a potent inhibitor of vascular SMC apoptosis. The antiapoptotic effect of CO is, in part, dependent on the activation of soluble guanylate cyclase and is associated with the inhibition of p53 expression and the suppression of cytochrome c release from the mitochondria.

Treatment of vascular SMC with a combination of inflammatory cytokines stimulates apoptosis in a time-dependent manner, as demonstrated by DNA laddering, positive annexin V staining, and caspase-3 activation. However, the exogenous administration of CO (50–200 ppm) exerts a potent antiapoptotic effect. These concentrations of exogenous CO have been demonstrated to be comparable to that produced by HO-1 activity in cultured cells and represents physiologically relevant concentrations [16,19]. Our finding that CO inhibits apoptosis in vascular SMC is consistent with recent reports obtained with murine fibroblasts and bovine aortic endothelial cells but it contrasts with other studies demonstrating that CO induces apoptosis in murine thymocytes and bovine pulmonary artery endothelium [19–22]. Furthermore, our observation that CO does not adversely affect vascular SMC viability is contrary to findings in bovine pulmonary artery endothelial cells where acute cytotoxic effects of CO have been reported [23]. Thus, CO may regulate apoptosis and cytotoxicity in a cell-specific manner. The mechanism by which CO inhibits apoptosis in vascular SMC appears to involve the activation of soluble guanylate cyclase since the soluble guanylate cyclase inhibitor ODQ partially reverses the antiapoptotic action of CO. The concentration of CO used in our study has been demonstrated to stimulate SMC soluble guanylate cyclase and lead to marked increases in the intracellular level of cGMP [16]. In addition, several studies have found that cGMP inhibits apoptosis [24–26]. Thus, CO-mediated increases in SMC cGMP production may contribute to the antiapoptotic effect of CO. In contrast, ODQ has no effect on the inhibition of apoptosis by CO in endothelial cells [19]. In endothelial cells, CO appears to block apoptosis by promoting the activation of p38 MAPK [19]. However, we found that CO does not augment the cytokine-mediated induction of p38 MAPK in vascular SMC. Moreover, the p38 MAPK inhibitor, SB203580, fails to reverse the antiapoptotic action of CO in SMC. Thus, distinct signaling pathways mediate the inhibition of apoptosis by CO in vascular endothelium and smooth muscle.

Since the release of cytochrome c from the mitochondria plays a central role in apoptosis, we examined whether CO regulates mitochondrial cytochrome c release. Indeed, we found that CO abrogates the cytokine-mediated translocation of cytochrome c from the mitochondria to the cytosol. The mechanism by which CO inhibits mitochondrial cytochrome c release is unknown. However, the capacity of CO to inhibit the expression of p53 may be significant. p53 is a well-characterized transcription factor that activates the mitochondrial death pathway in both a transcriptional-dependent and independent manner. p53 induces the transcription of numerous proteins, such as Bax, NOXA, PUMA, and p53AIP1, that localize to the mitochondria and trigger the liberation of cytochrome c [27]. In addition, recent studies indicate that p53 can directly interact with the mitochondria and stimulate cytochrome c release [28–30]. Thus, the ability of CO to inhibit the expression of p53 may prevent the release of cytochrome c from the mitochondria. The capacity of CO to block an integral step in the apoptotic pathway may also contribute to its ability to block apoptosis in response to a wide spectrum of proapoptotic agents [19,20].

The physiological relevance of our study was further underscored by the capacity of endogenously formed CO to likewise block SMC apoptosis. Treatment of SMC with inflammatory cytokines stimulates HO-1 protein expression in a time-dependent manner. However, inhibition of HO-1 activity by ZnPP-IX induces apoptosis as early as 24 h following cytokine exposure, suggesting that HO-1 delays the apoptotic response to cytokines. The antiapoptotic effect of HO-1 is likely mediated via the release of CO since the CO scavenger, hemoglobin, reverses the cytoprotection afforded by HO-1. Moreover, the other HO-1 products, biliverdin, bilirubin, and free iron, fail to inhibit cytokine-stimulated apoptosis. The inability of endogenous CO to prevent apoptosis following 48 h of cytokine treatment may arise from a decrease in CO synthesis at this time. Consistent with this suggestion, we found that the cytokine-mediated increase in HO-1 protein is reduced by over 40% after 48 h of cytokine stimulation relative to the levels observed following 24 h of exposure.

Since treatment of vascular SMC with inflammatory cytokines also induces the expression of inducible NO synthase [15,31], we examined whether NO is able to regulate SMC apoptosis. Consistent with an earlier study [32], we found that inhibition of inducible NO synthase activity attenuates apoptosis, indicating that endogenously released NO contributes to the apoptotic effect of the inflammatory cytokines. The downstream mediators of NO-induced apoptosis are not completely known but include the induction of p53, Fas, and Bax, as well as the inhibition of Bcl-2 expression (see Ref. [33]). Thus, at sites of vascular inflammation, the diatomic gases CO and NO exert divergent regulatory effects on SMC apoptosis. Interestingly, in an earlier study [34], we found that the induction of HO-1 by these inflammatory cytokines is dependent on the formation of NO, suggesting that the HO-1-catalyzed release of CO may function in a negative feedback fashion to limit the extent of SMC apoptosis.

Interestingly, studies from our laboratory and others suggest that high level HO-1 expression following adenoviral-mediated gene transfer induces SMC apoptosis [35,36]. Under these conditions, the proapoptotic action of HO-1 may be mediated via the generation of biliverdin and bilirubin, which at high concentration are known inducers of apoptosis [35,37]. Reversal of HO-1 related cytoprotection with increased expression following HO-1 transfection has also been demonstrated in fibroblasts and suggests caution when using genetic approaches to overexpress HO-1 [38].

The ability of CO to inhibit vascular SMC apoptosis may play an important pathophysiological role in the vasculature. By blocking SMC apoptosis, CO may increase the number of SMC in vascular lesions and thereby contribute to plaque development. However, apoptosis has also been implicated in plaque progression through the development of the acellular lipid necrotic core [14]. In addition, apoptosis of SMC has been detected in the vulnerable shoulder region of plaques, suggesting that this process may contribute to plaque destabilization and rupture [11]. The exposure of phosphatidylserine at the surface of apoptotic SMC also augments the procoagulant potential of the vessel wall by promoting the surface assembly and catalytic efficiency of the enzymes involved in the coagulation cascade leading to the generation of thrombin [39]. Thus, the capacity of HO-1-derived CO to block SMC apoptosis may play a critical role in regulating plaque progression and erosion and thrombosis. Since activated immune cells also contribute to plaque activation [40], the finding that CO induces apoptosis in thymocytes [21] may provide an additional mechanism by which this gas modulates plaque progression. The potential importance of the HO-1/CO system in promoting vascular homeostasis is supported by recent studies demonstrating that the induction of HO-1 activity attenuates vascular lesion formation in a mouse model of atherosclerosis and prevents thrombosis of coronary arteries during chronic exposure to hypoxia [41,42].

In conclusion, the present study demonstrates that CO inhibits vascular SMC apoptosis by blocking the expression of p53 protein and the release of cytochrome c from the mitochondria. The ability of HO-1-derived CO to block SMC apoptosis may play a fundamental role in reducing lesion formation at sites of vascular injury. Strategies aimed at delivering CO to sites of vascular injury may represent a potentially important therapeutic approach in treating vascular disease.

Time for primary review 30 days.

	Acknowledgements

 
This work was supported in part by the National Heart, Lung, and Blood Institute grants HL59976, HL36045, and HL62467. W. Durante is an Established Investigator of the American Heart Association.

	References

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

 

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