Published online before print July 13, 2006, doi: 10.1161/01.ATV.0000236204.37119.8d
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© 2006 American Heart Association, Inc.
Vascular Biology |
Protection of Human Vascular Smooth Muscle Cells From H2O2-Induced Apoptosis Through Functional Codependence Between HO-1 and AKT
From the Departments of Physiology (K.R.B., K.K.F., G.K., C.A.W., L.G.M.) and Anatomy and Cell Biology (M.Y.T., S.C.P.), Queen’s University, Kingston Ontario, Canada.
Correspondence to Dr Luis G. Melo, Department of Physiology, Queen’s University, 18 Stuart St, Kingston, Ontario, K7L 4S7. Canada. E-mail melol{at}post.queensu.ca
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Abstract |
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Objective— Oxidative stress (OS) induces smooth muscle cell apoptosis in the atherosclerotic plaque, leading to plaque instability and rupture. Heme oxygenase-1 (HO-1) exerts cytoprotective effects in the vessel wall. Recent evidence suggests that PKB/Akt may modulate HO-1 activity. This study examined the role of Akt in mediating the cytoprotective effects of HO-1 in OS-induced apoptosis of human aortic smooth muscle cells (HASMCs).
Methods and Results— HASMCs were transduced with retroviral vectors expressing HO-1, Akt, or GFP and exposed to H2O2. Cell viability was assessed by MTT assay. OS was determined by CM-H2DCFDA fluorescence, and apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), caspase-3 activity, and Bcl-2/Bad levels. Mitochondrial membrane potential (
m) was assessed by fluorescence-activated cell sorter (FACS) using JC-1. HO-1 reduced H2O2-induced OS and apoptosis. Akt knockdown removed the protective effect of HO-1 on m during exposure to H2O2. Conversely, HO-1 knockdown removed the protective effect of Akt on m. Inhibition of PI3K-Akt reduced induction of HO-1 protein expression by H2O2 and blocked its anti-apoptotic effects. The Akt-mediated upregulation of HO-1 was dependent on activation of HO-1 promoter by Nrf2.
Conclusion— HO-1 and Akt exert codependent cytoprotective effects against OS-induced apoptosis in HASMCs. These findings may have implications for the design of novel therapeutic strategies for plaque stabilization.
Oxidative stress induces smooth muscle cell apoptosis in the atherosclerotic plaque, leading to plaque instability. Heme oxygenase-1 (HO-1) exerts anti-oxidant, anti-inflammatory, and anti-apoptotic effects in the vessel wall. Here we report that the cytoprotective effect of HO-1 against pro-oxidant–induced apoptosis is mediated through codependent interaction with the survival gene Akt.
Key Words: apoptosis • flow cytometry • mitochondrial membrane potential • oxidative stress • vascular smooth muscle cells
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Introduction |
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Oxidative stress (OS) has been implicated in vascular injury leading to atherosclerosis, hypertension, restenosis, and vasospasm.1 Pro-oxidant species such as hydrogen peroxide (H2O2) exert dose-dependent effects on vascular cells, including cell proliferation, activation, and apoptosis.2 In the atherosclerotic plaque, excessive production of pro-oxidant species induces apoptosis of vascular smooth muscle cells in the fibrous cap, resulting in plaque instability.3–6 Because vascular smooth muscle cell (VSMC) loss precipitates plaque rupture and thrombosis,7–10 the protection of VSMC from apoptosis in the plaque has become an important therapeutic target for plaque stabilization.11,12
Heme oxygenase-1 (HO-1) is the rate-limiting enzyme involved in the conversion of heme into biliverdin, carbon monoxide, and free iron.13 The byproducts of heme breakdown have pleiotropic cytoprotective effects on the vessel wall.13,14 Bilirubin is a powerful antioxidant15 and carbon monoxide exerts vasodilatory, anti-inflammatory, anti-mitogenic, and anti-apoptotic effects in VSMCs and endothelial cells.16–19 The protective effects of HO-1 on VSMCs may be particularly important for maintenance of atherosclerotic plaque stability. Because of its anti-inflammatory and anti-apoptotic effects, HO-1 may reduce loss of VSMCs in the fibrous cap, and prevent plaque erosion and rupture. Indeed, several studies support the notion that HO-1 exerts an essential protective role in the vessel wall during atherogenesis.20 For example, HO-1 is upregulated in atherosclerotic plaques,21 suggesting that the increase in HO-1 gene expression may be a cytoprotective response to the oxidative and inflammatory microenvironment in the plaque. This is further supported by Yet et al,22 who reported that the absence of HO-1 exacerbates atherosclerotic lesion formation in apoE–/– mice. Others have shown that HO-1 over-expression markedly reduces atherosclerotic lesion formation and thrombosis.23–27
The mechanism underlying the protection of VSMC by HO-1 from oxidative stress- induced apoptosis is not known. Carbon monoxide has been reported to mediate the anti-apoptotic effects of HO-1 in response to inflammatory cytokine stimulation in VSMCs,17 but its role in protecting VSMCs from pro-oxidant–induced apoptosis has not been established. Paradoxically, one study reported increased apoptosis in rat VSMCs after exogenous overexpression of HO-1,28 suggesting that HO-1 may exert different dose-dependent effects on cell survival.29 More recently, several studies suggested that PI3K through the survival gene Akt may play a role in the induction of HO-1 gene expression and its anti-apoptotic effects in the presence of cellular stress.30,31 In addition, Akt also phosphorylates HO-1,32 suggesting a role of Akt in post-translational regulation of HO-1 activity. More significantly, simvastatin inhibits VSMC activation and proliferation by inducing HO-1 expression in an Akt-dependent manner.33 However, despite these findings, a functional dependence between Akt and HO-1 in protection of VSMCs from OS-induced apoptosis has not been established. Such a mechanism could have potential therapeutic implications, given the role of HO-1 and Akt in vascular homeostasis.14,34
Thus, in this study we examined the role of Akt activation in mediating the cytoprotective effects of HO-1 in pro-oxidant induced apoptosis in HASMCs.
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Methods |
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For more details, please see online Methods supplement at http://atvb.ahajournals.org.
Statistical Analysis
All results are presented as means±SE unless stated otherwise. Two-way analysis of variance (ANOVA) was used to compare combined and separate effects of time and treatment on HO-1 protein expression. One-way ANOVA coupled to Bonferroni multiple comparison post-hoc test was used to compare the effects of different treatments on CM-H2DCFDA fluorescence, cell viability, and apoptosis. Unpaired 2-tailed t test was used to compare differences in caspase-3 activity between HO-1 and GFP-transduced cells. P<0.05 was considered to indicate statistically significant difference.
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Results |
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HO-1 Overexpression Protects HASMCs Against Oxidative Stress, Maintains Cellular Viability, and Reduces Apoptosis
The effect of HO-1 overexpression in H2O2-induced OS and apoptosis in HASMC is shown in Figure 1. Transduction efficiency of HASMC by MSCV retrovirus was >90% after 2 rounds of transduction with 5 multiplicities of infection, and resulted in &2.5-fold increase in HO-1 protein levels compared with MSCV-GFP–transduced cells (supplemental Figure I, available at http://atvb.ahajournals.org). H2O2 increased OS, the number of TUNEL-positive cells, and reduced cellular viability in a dose-dependent manner 24 hours after exposure (supplemental Figure II). H2O2 increased OS significantly in GFP-transduced cells as measured by CM-H2DCFDA (Figure 1A). H2O2-induced OS was significantly reduced in HO-1 overexpressing cells compared with the GFP cells (Figure 1B). The increase in OS in GFP cells was accompanied by a &60% increase in TUNEL positive cells (Figure 1C, 1E, 1G). Concomitant with the decrease in OS, there was a significant reduction in the number of TUNEL-positive nuclei (Figure 1D, 1F, 1G) and caspase-3 activity (Figure 1H) and a parallel increase in overall cell viability (Figure 1I) in HO-1–transduced cells. Even at supra-physiological doses (>300 µmol/L) capable of causing >90% cell death in the control cells, HO-1 reduced apoptosis by >80%. (Figure 1G). The cytoprotective effect of HO-1 was accompanied by a time-dependent increase in the level of the anti-apoptotic protein Bcl-2 and a decrease in the level of the pro-apoptotic protein Bad (Figure 1J).
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The Cytoprotective Effect of Exogenous HO-1 Over-expression Against Oxidative Stress-Induced Apoptosis Is Dependent on Akt Activity
We postulated that the anti-apoptotic effect of HO-1 against OS may be mediated, at least in part, by a positive feedback interaction with the PI3K-Akt survival pathway. Figure 2 shows the effect of Akt on HO-1 protein expression and apoptotic cell death after exposure to H2O2 in GFP and HO-1–transduced cells. HO-1 protein expression increased time dependently up to 12 hours after exposure to 300 µmol/L of H2O2 in both the GFP and HO-1–transduced cells (Figure 2A, 2B; see also supplemental Figure III). As expected, the amount of HO-1 protein at any one time point was higher in HO-1–transduced cells than in the GFP-transduced cells. Inhibition of PI3K with LY294002 reduced HO-1 protein expression significantly in both the GFP and HO-1–transduced cells (Figure 2A, 2B) and further increased H2O2-induced apoptosis in GFP transduced cells (Figure 2C). LY294002 did not increase apoptosis in the HO-1 transduced cells in response to 300 µmol/L H2O2 (Figure 2D). However, at 600 µmol/L H2O2, the anti-apoptotic effect of HO-1 overexpression was completely removed by LY294002. To further define the potential interaction between HO-1 and Akt in cellular protection against OS-induced apoptosis, we used small interfering RNA oligonucleotides (siRNA) for human HO-1 and Akt 1/2. Transfection of HASMCs with fluorescein-conjugated scrambled sequences showed high levels of siRNA transfection efficiency as confirmed by intense green fluorescence in the cytosol, leading to marked decrease in protein expression (supplemental Figure IV). Interestingly, pretreatment with Akt 1/2 siRNA reduced the cytoprotective effects of HO-1 even at 300 µmol/L of H2O2 (Figure 2E), suggesting that a PI3K-independent mechanism(s) may contribute to the modulatory effects of Akt in HO-1-mediated cytoprotection in HASMC.
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HO-1 and Akt Exert Reciprocal Effects in Preservation of Mitochondrial Membrane Potential
To determine whether there were reciprocal effects of HO-1 and Akt in the cytoprotective response to H2O2, we used FACS analysis to assess changes in fluorescence of JC-1, a potentiometer dye that detects changes in mitochondrial membrane potential (m). Hyperpolarized intact mitochondria concentrate JC-1 in the intermembrane space resulting in JC-1 aggregation and fluorescence in the red spectrum (FL2). Depolarization caused by pore formation results in JC-1 aggregate release and dissociation to its monomeric form, which fluoresces in the green spectrum (FL1). Cells that are healthy are most intense for the red aggregate and localize to the third log (Figure 3A, 3J), whereas cells with perforated mitochondria shift to the lower logs (101 to 102), and dead cells to the lattermost log (100). In comparison to control cells (Figure 3A) we observed a large decrease in m in HASMCs exposed to H2O2 (Figure 3B, 3J) Transfection with a scrambled siRNA had no detrimental effect on m (Figure 3C, 3J). HO-1 overexpression attenuated mitochondrial depolarization (Figure 3D, 3J) as indicated by a higher percentage of cells in the third decade and fewer cells in the lower decades. However, inhibition of Akt with siRNA removed the protective effect of HO-1 over-expression on m (Figure 3E, 3J). This effect was recapitulated when HO-1 siRNA was used in HO-1–overexpressing cells (Figure 3F, 3J). m was preserved in Akt-overexpressing cells exposed to H2O2 (Figure 3G, 3J). The cytoprotective effect of Akt on m was attenuated by targeting HO-1 with siRNA (Figure 3H, 3J). The deleterious effect of H2O2 on m was recapitulated by siRNA targeting Akt in Akt-overexpressing cells (Figure 3I, 3J).
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HO-1 and Akt Exert Reciprocal Effects on Other Protein Levels
To understand the potential interaction between HO-1 and Akt on cellular protection against OS-induced apoptosis, we used pharmacological inhibitors of PI3K and siRNA for human HO-1 and Akt 1/2 (for transfection and gene knockdown efficiency (supplemental Figure IV) to determine the reciprocal effects of HO-1 and Akt on each other’s protein levels. HO-1 overexpression increased Akt phosphorylation by 70% to 80% relative to GFP control cells in response to H2O2 without affecting the total Akt protein levels (Figure 4A). Inhibition of PI3K-Akt by LY294002 markedly reduced HO-1 protein levels in both HO-1 and GFP-transduced cells (Figure 4B). This was further confirmed using gene knockdown with siRNA (Figure 4C). HO-1 siRNA reduced H2O2 induced Akt phosphorylation by &30% relative to control cells transfected with a scrambled sequence. Reciprocally, Akt knockdown with siRNA nearly suppressed HO-1 protein expression in response to H2O2 (Figure 4C), thus indicating that Akt and HO-1 reciprocally stimulate each other’s activity in a codependent manner. Akt siRNA markedly reduced steady state HO-1 mRNA levels after exposure to 300 µmol/L H2O2, suggesting that Akt regulates HO-1 expression by a transcriptional mechanism (Figure 4D).
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Akt Increases HO-1 Levels Via Cap’n Collar Transcription Factor Nrf2
We investigated the mechanism underlying the stimulation of HO-1 by Akt. Exposure to 300 µmol/L H2O2 increased HO-1 promoter activity in a time-dependent fashion, peaking at 3 hours (Figure 5A). The increase in HO-1 promoter activity was preceded by an increase in Akt phosphorylation (Figure 5B) and coincided with increased Nrf2 protein levels (Figure 5B). The transcription factor appeared diffuse and exclusively localized to the cytosol in unstimulated conditions (Figure 5C to 5F). On exposure to H2O2, Nrf2 concentrated in the perinuclear region and translocated to the nucleus (Figure 5G to 5N), in parallel with the increased promoter activity (Figure 5A). The translocation of Nrf2 peaked at 3 to 6 hours after H2O2 and preceded the induction of HO-1 (Figure 5B, 5G to 5J), which declined steadily thereafter (Figure 5B, 5K to 5N).
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To determine the role of Nrf2 in mediating the effect of Akt in H2O2-induced HO-1 expression, we treated cells with Akt or Nrf2 siRNA. Akt knockdown was associated with reduced Nrf2 and HO-1 expression, compared with cells treated with scrambled siRNA (Figure 6A). This was accompanied by reduced HO-1 promoter activity (Figure 6B). Similarly, Nrf2 knockdown decreased HO-1 protein expression (Figure 6A) and promoter activity (Figure 6B). Immunohistochemical analysis of Nrf2 localization showed that both Akti and Nrf2i markedly reduced H2O2-induced perinuclear localization and nuclear translocation of Nrf2 (Figure 6G to 6J) compared with untreated (Figure 6C, 6D) or scrambled siRNA-treated cells (Figure 6E, 6F).
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Discussion |
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Plaque rupture rather than luminal stenosis is the most severe clinical manifestation of advanced atherosclerosis, leading to thrombosis and potentially fatal acute coronary events.11 Vascular smooth muscle apoptosis occurs throughout atherogenesis, but is accentuated in advanced lesions because of the heightened inflammatory and pro-oxidant micro-environment of the plaque.7,9 This poses a problem for plaque stability because VSMCs synthesize the extracellular matrix components required for tensile strength of the fibrous cap. Thus, the development of protective strategies for prevention of VSMC apoptosis is an important therapeutic target to achieve atherosclerotic plaque stabilization. Heme oxygenase-1 and Akt activity are obligatory protective mechanisms in the vessel wall, and their anti-apoptotic properties may be essential to achieve plaque stabilization. Here we show for the first time to our knowledge that exogenous overexpression of HO-1 confers marked protection from pro-oxidant induced apoptosis in human aortic VSMCs. More importantly, our results indicate that the cytoprotective effect of HO-1 against oxidative stress induced cell death in HASMC is, at least partly, dependent on Akt, suggesting that these 2 cytoprotective enzyme systems function cooperatively to inhibit pro-oxidant induced apoptosis of HASMCs. We believe that this functional interaction may have implications for the design of new therapeutic strategies for plaque stabilization in atherosclerosis.
HO-1 exerts pleiotropic effects in the vessel wall, including the inhibition of apoptosis, proliferation, inflammation and adhesion molecule expression in VSMC in culture and in injured arteries.14,16–18,35 Furthermore, HO-1 is upregulated in atherosclerotic plaques,21 and HO-1 overexpression reduces atherosclerotic lesions in genetically pre-disposed animals.26,27 Thus, these findings indicate that HO-1 is an important anti-atherogenic agent. The dual inhibitory effects of HO-1 on VSMC proliferation and apoptosis may be particularly important in atherogenesis. These effects may act to limit cell replication and excessive luminal occlusion in the developing lesion and prevent excessive apoptosis in the advanced lesion. In this context, our current findings suggest that exogenous HO-1 supplementation may be a useful therapeutic strategy for protection of VSMCs in the pro-inflammatory and pro-oxidant milieu of the advanced atherosclerotic lesion.
The mechanism by which HO-1 inhibits apoptosis in VSMC is not fully understood. Our current results indicate that the protective effect of HO-1 against pro-oxidant–induced apoptosis in HASMCs is critically dependent on Akt activity. Furthermore, the cytoprotective effect of Akt appears to be, at least partially, dependent on HO-1 activity, suggesting that these 2 enzymes function in a codependent and cooperative fashion to confer protection from OS in HASMCs. This premise is supported by our results showing that inhibition of Akt activity markedly reduces the ability of HO-1 to inhibit apoptosis and preserve mitochondrial membrane potential. Indeed, pretreatment of cells with Akt siRNA led to almost complete knockdown of HO-1 promoter activity, mRNA, and protein expression, indicating that the role of Akt in HO-1 mediated cytoprotection may be caused by its ability to promote HO-1 transcription.
The mechanism linking exogenous H2O2 to Akt activity and induction of HO-1 is not known. HO-1 levels are primarily regulated at the transcriptional level by a number of redox sensitive transcription factors.36,37 H2O2 diffuses freely across the cell membrane and activates intracellular signaling molecules that may converge to induce HO-1 gene transcription via stimulation of redox-sensitive transcription factors such as NF-
B, AP-1, and Nrf-2.38–42 Our data indicate that the effect of Akt on HO-1 levels occurs primarily at the level of transcription, because Akt inhibition markedly reduces HO-1 promoter activity and steady state mRNA levels. This is in agreement with the data reported by Salinas et al30 on PC12 cells. However, our data show that Akt siRNA decreases Nrf2 perinuclear localization and nuclear translocation in response H2O2. Furthermore, HO-1 promoter activity is comparably inhibited by Akt and Nrf2 siRNA, suggesting that the effect of Akt on H2O2-induced HO-1 transcriptional activation is, at least in part, mediated via Nrf2. In this regard, we note that Nrf2 has been reported to play an essential role in induction of HO-1 in response to hemin43 and the anti-oxidant carnosol by a mechanism that is dependent on upstream activation by PI3K/Akt.44 Furthermore, PI3K/Akt regulates the nuclear translocation of Nrf2 in response to oxidative stress.45 In addition, Akt and HO-1 may also interact at the post-translational level. Akt phosphorylates HO-1 at serine 188 both in vitro and in vivo, resulting in a modest increase in HO activity.32 Interestingly, our data show that HO-1 overexpression results in increased levels of phosphorylated Akt without affecting total Akt. It is not clear from our results whether HO-1 directly phosphorylates Akt or whether HO-1 inhibits Akt dephosphorylation by reducing OS.46 This suggests that Akt and HO-1 may operate in a positive feedback mechanism, whereby the level of HO-1 expression reciprocally augments Akt activation, which in turn increases HO-1 expression.
The current findings may have therapeutic implications for atherosclerosis. A recent study reported that simvastatin markedly induced HO-1 and inhibited proliferation and inflammation-mediated activation in vascular smooth muscle cells in vitro and in the medial layer of blood vessels.33 Interestingly, these pleiotropic effects of simvastatin were found to be dependent on p38 and PI3K-Akt. Our current results show that HO-1–mediated protection of HASMCs from oxidative stress induced apoptosis is dependent on Akt activity. A plausible working model for the interaction between Akt and HO-1 in cytoprotection from oxidative stress may involve activation of Akt by H2O2 either directly or proximally at the level of PI3K (supplemental Figure V). Activated Akt may act as a relay to phosphorylate Nrf2, promoting its dissociation from the cytosolic repressor Keap1 and its translocation into the nucleus, where it induces HO-1 gene transcription by binding to the antioxidant response element (ARE) in the HO-1 promoter. A positive feedback loop between Akt and HO-1 driven by reactive oxygen species may operate at the post-translational level by reciprocal phosphorylation events between these 2 enzymes. Akt activity may be further enhanced via BVR-mediated phosphorylation. Termination of the positive feedback loop between Akt and HO-1 is likely mediated by bilirubin, which may buffer cytosolic ROS accumulation. However, confirmation of this potential mechanism of cytoprotection remains to be established.
In conclusion, our results reveal for the first time to our knowledge a functional codependence between HO-1 and Akt in mediating cytoprotection against oxidative stress induced cell death. Given the prevalence of oxidative stress and apoptosis in advanced atherosclerotic disease, this novel interaction between 2 key cytoprotective systems may provide the rationale for the development of therapeutic strategies for plaque stabilization and prevention of plaque rupture and thrombosis.
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Acknowledgments |
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Sources of Funding
This work was supported by grants from the Canadian Institutes of Health Research (CIHR, MOP 60500) and the Heart and Stroke Foundation of Ontario (HSFO, NS 5029) to Dr Melo. Dr Pang is supported by a grant (T-5326) from the HSFO. Dr Ward is supported by grants from CIHR (MOP 77792) and HSFO (T-5143). Keith Brunt is supported by a doctoral award from CIHR (GREAT Training Program). Dr Melo is Canada Research Chair in Molecular Cardiology and a New Investigator of the Heart and Stroke Foundation of Canada.
Disclosures
None.
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Footnotes |
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Original received May 4, 2006; final version accepted June 21, 2006.
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