New Partners in Atherosclerotic Progression?
See related article, pages 42–50
Heme, Fe-protoporphyrin IX, is an essential building block for oxygen carrying hemoglobin and myoglobin. Physiologic recycling of heme occurs in many tissues, predominantly in the reticuloendothelial system, and is dependent on hemoxygenase-1 (HO-1). Heme is degraded by HO-1 into constituent carbon monoxide (CO), Fe2+, and biliverdin and then converted to bilirubin. CO and biliverdin possess beneficial attributes through signaling and antioxidative capacities, respectively, whereas Fe2+ is a strong oxidant. The oxidative capacity of Fe2+ is contained by ferritin, a Fe2+-binding protein that serves as a chaperone transporting Fe2+ for synthesis of iron containing proteins. The importance of heme metabolism has been highlighted in clinical observations in which the lack of capacity to degrade heme observed in a 6-year-old child with HO-1 deficiency was associated with plaque formation in the aorta and hyperlipidemia,1 and low HO-1 expression is correlated with increased cardiovascular risk. These findings are strongly supported by studies in mice lacking HO-1.2 Thus it is clear that lack of HO-1 is detrimental in the cardiovascular system; however, one must consider whether the increased risk is attributable to the accumulation of heme or the consequences of decreased heme degradation products?
Changes in vascular homeostasis with disease or injury inevitably lead to increased oxidative stress. This in part is a consequence of elevated local and circulating heme. Unbound heme, is a strong oxidant, and its hydrophobic nature permits rapid entry into cells of the vascular wall. Once heme enters the lipid bilayer within the cell, it is readily capable of increasing oxidative stress to levels that can significantly exceed the antioxidant mechanisms and capacity of the cell. Thus vascular disease such as atherosclerosis and injury resulting in thrombus formation are sources of increased free heme and oxidative stress that can overwhelm cellular defenses and exacerbate established injury and disease. Increased oxidative stress triggers responses that counter balance the consequences of increased oxidative stress on protein stability, such as increased expression of HO-1, ferritin, thioredoxin, heat shock protein 70, and c-fos and, conversely, responses that lead to progression of vascular pathology notably increased oxidation of LDLs, cross-linking of apolipoprotein B100, and smooth muscle proliferation.3
The study by Hasan and Schafer in this issue of Circulation Research4 draws attention to early mechanisms that are activated in vascular smooth muscle (VSM) in response to hemin (oxidized heme) exposure and how they may contribute to the confounding actions of heme. Their findings are significant because they demonstrate a novel mechanism that implicates heme-dependent oxidative stress in atherosclerotic progression. Initial responses to heme are aimed at the preservation of the vascular compartment. As noted above, the acute response includes increased expression of factors that minimize oxidative damage to the cell by scavenging excess reactive oxygen species (ROS) and preserve protein and cellular function. Hasan and Schafer have found that the early growth response 1 (Egr-1) gene is rapidly upregulated with hemin (Figure, top). This is a highly relevant finding in the context of vascular disease. Egr-1 has been found to respond to numerous stressors and injury and, to a greater extent, has been associated with a progression of vascular dysfunction.5 Thus identification of hemin as a contributing factor is an important finding and contributes to the further understanding of atherosclerosis etiology. To date, there has been little suggestion of a role for heme in progression of plaque formation or vulnerability. Induction of Egr-1 has been associated with angiogenesis and thus hemin may also contribute as microvessel density is increased in advanced plaques. However, in contrast to Egr-1 proliferative actions, the authors have found the action of hemin on VSM proliferation to be inhibitory, underscoring the complex nature of heme actions.
Hasan and Schafer demonstrate that hemin-induced Egr-1 expression is derived solely from its ability to induce ROS. Using a panel of ROS scavengers and inhibitors, the authors show that superoxide is necessary for Egr-1 induction because the superoxide scavenger, tiron, completely inhibits hemin-induced ROS. Inhibition of NADPH oxidase activity with apocynin also completely abolishes hemin-induced ROS. Confirmation of these results using alternate approaches such as genetically targeted models or small interfering RNA knockdown will further strengthen the role of NADPH oxidase. These results are significant because they demonstrate that increased ROS levels may be a consequence of the activation of NADPH oxidase by heme and not the result of Fe2+ toxicity. This suggests that heme remains intact in VSM under experimental conditions, eliminating a role for HO-1 and other degradation products, CO and biliverdin/bilirubin, and also excludes the nonenzymatic oxidative degradation of heme and the Fenton reaction in ROS production. This also implies that insufficient HO-1 in atherosclerosis may create similar conditions that facilitate induction of Egr-1.
Hemin-induced ROS activation of ERK leading to Egr-1 expression, although not examined by Hasan and Schafer, is an important aspect of this study and should be noted. Of interest is the transduction of ROS signal to ERK activation potentially through Ras. Ras has been shown to be activated in a redox-sensitive manner by the actions of glutaredoxin 1 and thioredoxin 1.6 Thioredoxin 1 can also activate nuclear factor (NF)-κB, which is also relevant to these studies. Another important potential link for heme-dependent activation is the unknown relationship between novel glutaredoxin 2 and 5, both of which have been associated with iron-catalyzed oxidative stress.7,8 The observation that ROS formation seems to be derived from NADPH oxidase activation and the subsequent increase in superoxide anion raises the important question regarding the mechanism by which NADPH oxidase is activated by hemin and whether this may be an ERK dependent event.
It is important to note here that Egr-1 induction with hemin is inhibited by CO (via the CO donor CORM-2), and thus HO-1 degradation of heme serves a negative-feedback function. This finding is important in the context of atherosclerosis, in which, as noted above, HO-1 levels may be insufficient to break down increased levels of heme. Heme is an important inducer of HO-1 expression, and thus low levels of induction would confound the effort of the cell to decrease heme levels and provide negative feedback via CO to heme-dependent signaling, both of which would contribute to prolonged Egr-1 elevation (Figure, bottom). Although this appears to be a confounding factor leading to atherosclerotic progression, this could be viewed as an opportunity to use strategies that would increase HO-1 expression and CO production to attenuate Egr-1 signaling. The authors also show that a target gene of Egr-1, NGF-1-A binding (NAB2), is upregulated with hemin. NAB2 is a transcriptional corepressor of Egr-1 and thus should serve a negative-feedback function to halt continued elevated transcription and expression of Egr-1 targets. The integrity of this feedback loop would be critical for appropriate attenuation of Egr-1 target gene expression, tissue factor (TF), and plasminogen activator inhibitor (PAI)-1 for example (Figure, top). NAB-2 repressor activity is dependent on promoter context and thus outcome will be determined for the specific Egr-1 target.
The authors have clearly delineated the transcriptional elements activated for Egr-1 transcription, providing strong mechanistic insight into hemin action (Figure, top). Extracellular signal-regulated kinase (ERK)-1–dependent Elk-1 was activated and translocated to the nucleus rapidly with hemin treatment. An Elk-1 collaborating factor, serum response factor (SRF), and NF-κB were also activated and found increased in the nucleus. Specific targeting of these factors to the human Egr-1 promoter was verified by chromatin immunoprecipitation analysis. Knockdown of Elk-1 and inhibition of NF-κB both prevented hemin induction of Egr-1 demonstrating their mutual dependence at the Egr-1 promoter.
Functional correlation with hemin-induced Egr-1 expression included analysis of 3 potential target genes involved in vascular hemostasis, TF and PAI-1, and the Egr-1 transcriptional corepressor NAB2. TF and PAI-1 are both implicated in the progression of atherosclerotic vascular disease. TF expression has been associated with VSM of the media and neointima after balloon injury and is found in several cell types in atherosclerotic plaque including VSM. The prothrombotic activity of TF thus can contribute to vascular pathology under conditions of acute arterial injury or plaque rupture in acute infarction or unstable angina. PAI-1 is also associated with neointima and plaque formation because of its ability to facilitate VSM proliferation and stabilize fibrin matrix, particularly relevant to thrombus formation and destabilization in the plaque core. Heme increased Egr-1 binding to Egr-1 motifs on TF, PAI-1, and NAB2 promoters by chromatin immunoprecipitation analysis, demonstrating direct interaction with target genes. With increased expression, TF and PAI-1 activity were increased. The increase in TF and PAI-1 activity over the relatively short exposure to hemin in this study demonstrates that Egr-1 expression in VSM has the capacity to impact vascular integrity and contribute to processes leading to plaque formation. Egr-1 has been shown to increase expression of growth factors, adhesion molecules, cytokines, and clotting factors that may also contribute to dysfunctional Egr-1 regulation, leading to prolonged expression (Figure, bottom).
Although all studies in this report were performed in cultured VSM with acute exposure to hemin, they lay the ground work to determine the etiology of heme induced oxidative stress on vascular injury in vivo. It is clear that Egr-1 levels are elevated in atherosclerotic tissues, a state that may be considered chronically elevated and this naturally raises questions regarding how attenuation of Egr-1 signaling is lost. This is of interest, as noted above, because hemin increases HO-1 and NAB2 expression, both of which provide negative feedback to attenuate this response. Could dysfunction of these negative feedback mechanisms be the cause of chronic elevated Erg-1 (Figure, bottom)? Some studies have associated low levels of HO-1 in patients with high cardiovascular risk, and these therefore raise the question regarding mechanisms responsible for increased HO-1 expression with stress and whether they are sufficient? Chronic exposure to heme, as may be the case after cap rupture, thrombus formation, and resorption, may serve to maintain increased ROS levels and induction of Egr-1 expression. Thus studies that address the effect of chronic vascular exposure to heme in the context of vascular injury and atherosclerotic disease should be anticipated to extend the significance and impact of these findings.
Sources of Funding
Work in the laboratory of the author is supported by NIH grant HL071896.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
Stocker R, Perrella MA. Heme oxygenase-1: a novel drug target for atherosclerotic diseases? Circulation. 2006; 114: 2178–2189.
Balla J, Vercellotti GM, Nath K, Yachie A, Nagy E, Eaton JW, Balla G. Haem, haem oxygenase and ferritin in vascular endothelial cell injury. Nephrol Dial Transplant. 2003; 18 (suppl 5): v8–v12.
Hasan RN, Schafer AI. Hemin upregulates Egr-1 expression in vascular smooth muscle cells via ROS–ERK-1/2–Elk-1 and NF-κB. Circ Res. 2008; 102: 42–50.
Khachigian LM. Early growth response-1 in cardiovascular pathobiology. Circ Res. 2006; 98: 186–191.
Kuster GM, Pimentel DR, Adachi T, Ido Y, Brenner DA, Cohen RA, Liao R, Siwik DA, Colucci WS. Alpha-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes is mediated via thioredoxin-1-sensitive oxidative modification of thiols on Ras. Circulation. 2005; 111: 1192–1198.
Lillig CH, Lonn ME, Enoksson M, Fernandes AP, Holmgren A. Short interfering RNA-mediated silencing of glutaredoxin 2 increases the sensitivity of HeLa cells toward doxorubicin and phenylarsine oxide. Proc Natl Acad Sci U S A. 2004; 101: 13227–13232.
Wingert RA, Galloway JL, Barut B, Foott H, Fraenkel P, Axe JL, Weber GJ, Dooley K, Davidson AJ, Schmid B, Paw BH, Shaw GC, Kingsley P, Palis J, Schubert H, Chen O, Kaplan J, Zon LI. Deficiency of glutaredoxin 5 reveals Fe-S clusters are required for vertebrate haem synthesis. Nature. 2005; 436: 1035–1039.