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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1483.)
© 2001 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Expression of Glutaredoxin in Human Coronary Arteries

Its Potential Role in Antioxidant Protection Against Atherosclerosis

Masanori Okuda; Nobutaka Inoue; Hiroshi Azumi; Tadashi Seno; Yoshihiko Sumi; Ken-ichi Hirata; Seinosuke Kawashima; Yoshitake Hayashi; Hiroshi Itoh; Junji Yodoi; Mitsuhiro Yokoyama

From the Division of Cardiovascular and Respiratory Medicine, Departments of Internal Medicine (M.O., N.I., T.S., Y.S., K.-i.H., S.K., M.Y.) and Surgical Pathology (H.A., Y.H., H.I.), Kobe University Graduate School of Medicine, Kobe, and the Department of Biological Response (J.Y.), Institute for Virus Research, Kyoto University, Kyoto, Japan.

Correspondence to Nobutaka Inoue, Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, chuo-ku, Kobe, 650-0017, Japan. E-mail nobutaka{at}med.kobe-u.ac.jp

	Abstract

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Abstract—— Oxidative stress is considered an important factor in atherogenesis. Mammalian cells have a complex network of antioxidants such as catalase, superoxide dismutase, and glutathione peroxidase. However, the mechanisms that regulate the cellular redox state in the vessel wall remain unclear. Recent study has shown that thioredoxin, a thiol-disulfide oxidoreductase, is expressed in atherosclerotic plaques of human carotid arteries. In this study, we investigated the localization and expressional change of glutaredoxin and thioredoxin, two important members of the thiol-disulfide oxidoreductases, in autopsy samples of human coronary arteries. In nonatherosclerotic coronary arteries, glutaredoxin was expressed in endothelial cells, in fibroblasts of the adventitia, and most intensely in medial smooth muscle cells. Interestingly, in atherosclerotic lesions such as hypercellular lesions, the infiltrating macrophages highly expressed glutaredoxin. The expressional pattern of thioredoxin was quite similar to that of glutaredoxin. Western blot analysis demonstrated that hydrogen peroxide stimulated the expression of glutaredoxin in a time- and dose-dependent manner in cultured human coronary artery smooth muscle cells. Fluorescence microtopography with dihydroethidium demonstrated that the generation of reactive oxygen species was associated with the expression of glutaredoxin. These results suggest the possible involvement of thiol-disulfide oxidoreductases in antioxidant protection in human coronary arteries.

Key Words: antioxidants • atherosclerosis • coronary artery disease • free radicals

	Introduction

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Oxidative stress induced by reactive oxygen species (ROS) is implicated in the pathogenesis of a variety of vascular diseases, including atherosclerosis, hypertension, and coronary artery disease.^1–3 The redox state is finely tuned to preserve cellular homeostasis through the expression and regulation of oxidant and antioxidant enzymes. Mammalian cells have a complex network of antioxidants such as catalase, superoxide dismutase, and glutathione peroxidase to scavenge ROS. In addition to these enzymes, the members of a family of thiol-disulfide oxidoreductases act as cytoprotective antioxidants. The thiol-disulfide oxidoreductases maintain the reduced thiol-disulfide status of the cytosol and are considered to regulate various cell functions, such as differentiation, gene transcription, cell growth, and apoptosis.^4–7 The two most important thiol-disulfide oxidoreductases are glutaredoxin (GRX) and thioredoxin (TRX).⁸

See cover

GRX, also known as thioltransferase, is a ubiquitously expressed, small cytosolic protein with a molecular mass of 11.5 kDa. GRX was discovered as a glutathione-dependent hydrogen donor for ribonucleotide reductase in a nonviable mutant of Escherichia coli lacking TRX.⁹ GRX protein displays a high degree of amino acid sequence homology among mammalian species.¹⁰ TRX and GRX are members of a superfamily of low-molecular-mass proteins that catalyze the reduction of disulfide bonds in a variety of proteins. GRX as well as TRX exerts its functions via disulfide exchange reaction by utilizing a Cys-Pro-Tyr-Cys active site. TRX coupled to NADPH and TRX reductase can reduce disulfides in many substrates, whereas GRX coupled to NADPH and glutathione reductase enables the monothiol reduced glutathione to reduce several disulfides (Figure I online; please see http://atvb.ahajournals.org). Previous investigations have indicated that the TRX/GRX system has a growing number of functions, such as modification of intracellular signal transduction, gene activation, and cytoprotection; however, the significance of these thiol-disulfide oxidoreductases in atherogenesis remains to be elucidated. Recently, Takagi et al¹¹ reported that TRX was expressed in endothelial cells (ECs) and macrophages in atheromatous plaques from human carotid arteries, suggesting that TRX may play an antioxidative role in atherosclerotic lesions, where oxidative stress is thought to be increased. In this study, we investigated the localization of TRX as well as of GRX by immunohistochemical study and double-labeling immunofluorescence in human coronary artery sections from autopsy cases. Furthermore, the role of ROS in their expression was examined by fluorescence microtopography with dihydroethidium.

	Methods

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Preparation of Human Tissue Specimen
Human coronary arteries were collected from 14 autopsy cases (nonatherosclerotic segments, n=11; atherosclerotic segments, n=24) from persons aged 47 to 83 within 5 hours after death. Coronary arteries were removed from the heart and cut into 3-mm-long segments. For immunohistochemistry and immunofluorescence examination, serial tissues were embedded in OCT^TM compound, snap-frozen in LN₂, and stored at -80°C.

Immunohistochemistry
Immunohistochemistry was performed as previously described.¹² In brief, the tissue slices were fixed with 100% acetone at -20°C for 10 minutes. The sections were blocked with carrier protein (Dako LSAB kit^TM, Dako A/S) for 30 minutes at room temperature and then incubated with a primary antibody overnight at 4°C. The primary antibodies were rabbit polyclonal antibody against a synthetic polypeptide of the C-terminal amino acids of human GRX and a mouse antibody against a synthetic polypeptide of the C-terminal amino acids of human TRX. Sections were washed with Tris-based buffer and then incubated with biotinylated goat anti-rabbit immunoglobulins (Dako), washed again in Tris-based buffer, and finally incubated with streptavidin–horseradish peroxidase conjugate (Dako LSAB kit^TM, Dako). For a negative control, the primary antibody was replaced with a rabbit nonspecific immunoglobulin.

Double-Labeling Immunofluorescence
To identify the types of GRX-expressing cells, double-labeling immunofluorescence was performed as previously described.¹³ The sections were blocked with a carrier protein (Dako LSAB kit^TM, Dako) for 30 minutes at room temperature and incubated with anti-human GRX antibody and antibody for cell-specific markers at the same time overnight at 4°C. These cell-specific antibodies were mouse monoclonal anti-human CD68 antibody (clone KP-1, Dako) for macrophages, anti-smooth muscle -actin antibody (clone 1A4, Dako) for smooth muscle cells (SMCs), anti–von Willebrand factor antibody (clone F8/86, Dako) for ECs, and anti–prolyl-4-hydroxylase antibody (clone 5B5, Dako) for fibroblasts. After a 15-minute washing procedure with 50 mmol/L Tris-based buffer, TRITC-conjugated swine anti-rabbit immunoglobulins (Dako) and FITC-conjugated sheep anti-mouse immunoglobulins (Amersham Pharmacia Biotech UK Ltd) were applied as secondary antibodies for 1 hour at room temperature. A laser scanning confocal imaging system (MRC-1024, Nippon Bio-Rad Laboratories) was used to examine these samples. The presence of GRX was demonstrated by red immunofluorescence labeling. The presence of the cell-specific markers was demonstrated by green immunofluorescence labeling. Yellow immunofluorescence labeling showed colocalization of red and green labeling.

Oxidative Fluorescence Microtopography
As previously described, dihydroethidium oxidative fluorescence dye was used to evaluate in situ production of ROS.^14,15 Unfixed frozen samples were cut into 20-µm-thick sections and placed on glass slides. Dihydroethidium (10 µmol/L) was applied to each tissue section, and then the sections were coverslipped. The slides were incubated in a light-protected humidified chamber at 37°C for 30 minutes. The image of dihydroethidium was obtained by a laser scanning confocal imaging system (MRC-1024) with a 585-nm long-pass filter. The immunoreactivity of GRX in serial sections was simultaneously examined with an immunofluorescence method by using rabbit polyclonal anti-GRX antibody and FITC-conjugated donkey anti-rabbit immunoglobulins (Amersham Pharmacia Biotech). The images of ROS generation and the immunoreactivity of GRX were demonstrated by red and green fluorescence, respectively.

Western Blot Analysis
Human coronary artery smooth muscle cells (CASMCs) were obtained from Clonetics (catalog No. CC-2583) and used at passages 4 to 8. Human CASMCs were cultured in a special medium (Clonetics, catalog No. CC-3181) supplemented with the manufacturer’s reagent (Clonetics, catalog No. CC-4148) until confluent on 60-mm dishes. After incubation with H₂O₂, the cultured cells were washed 2 times with ice-cold PBS, and they were gently scraped with lysis buffer containing protease inhibitors (50 mmol/L Tris/HCl [pH 7.4], 1 mmol/L EDTA, 500 µmol/L PMSF, 2 µmol/L leupeptin, and 10 µg/mL aprotinin). The cell suspension was homogenized in an ultrasonicator (15 seconds, 4 times) on ice and cleared by centrifugation. Equal amounts of protein (50 µg protein per lane), estimated by the method of Bradford (Bio-Rad), was electrophoresed on a 15% SDS-polyacrylamide gel and then electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore^TM). The membrane was blocked with buffer A (10 mmol/L Tris/HCl [pH 7.4], 100 mmol/L NaCl, and 0.1% Tween-20^TM) containing 1% skim milk at room temperature for 3 hours and incubated with primary antibodies at room temperature overnight. The membrane was then incubated with horseradish peroxidase–labeled donkey secondary antibodies (Amersham) at room temperature for 1 hour. The signals were detected with an enhanced chemiluminescence method (Amersham). Analysis of densitometry was performed with NIH Image software, version 1.60. Cell viability after treatment with H₂O₂ was >97% by the trypan blue exclusion test. Under our experimental conditions, cytotoxic effects were not observed.

	Results

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GRX Expression in Human Coronary Arteries
All sections were examined by hematoxylin-eosin staining and classified as nonatherosclerotic (n=11) or atherosclerotic (n=24) coronary arteries according to the reports of the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association.^16–18 In nonatherosclerotic coronary arteries, there was only mild and diffuse adaptive intimal thickening. In atherosclerotic segments, various pathological changes, including fatty streaks, accumulation of lipid, intimal disorganization, and infiltration of inflammatory cells, were observed.

The localization of GRX in human coronary arteries was examined by immunohistochemical study with an anti-human GRX antibody. Sections of coronary arteries taken from nonatherosclerotic coronary arteries showed that GRX was expressed most intensely in the media and was also observed in the endothelium and adventitia (Figure 1A). When the primary antibody was replaced with a rabbit nonspecific immunoglobulin, no reactivity was detected (data not shown). In atherosclerotic coronary arteries, the progression of atherosclerosis did not yield an additional increase in GRX expression in the endothelium, media, and adventitia. However, the inflammatory cells, which infiltrated into the fibrous plaques and hypercellular lesions, were strongly positive for GRX (Figures 1B and 1C).

View larger version (65K): [in this window] [in a new window]   Figure 1. Immunohistochemical analysis of GRX in human nonatherosclerotic (A) and atherosclerotic (B and C) coronary arteries. Immunoreactivity of GRX was observed most intensely in the media and was also observed in the endothelium and adventitia of nonatherosclerotic specimens. In addition to the endothelium and media, intense staining by GRX in infiltrating inflammatory cells was observed in atherosclerotic coronary arteries (B and C). Sections shown are representative of 35 experiments. Original magnification: A and B, x200; C, x400.

Characterization of GRX-Expressing Cells
To identify the types of GRX-expressing cells, double-labeling immunofluorescence with an anti-GRX antibody and cell-specific antibodies was performed (Figure 2). In atherosclerotic coronary arteries, GRX-expressing cells in the endothelium, media, and adventitia were positive for von Willebrand factor (A, E, and I in Figure 2), -actin (B, F, and J in Figure 2), and prolyl-4-hydroxylase (C, G, and K in Figure 2), respectively. In the neointima, the majority of GRX-expressing cells were CD68-positive macrophages (D, H, and L in Figure 2), and the others were -actin–positive SMCs (data not shown).

View larger version (41K): [in this window] [in a new window]   Figure 2. Double-labeling immunofluorescence to identify the types of GRX-expressing cells. Upper panels (red) show immunofluorescence of GRX (A through D). Middle panels (green) show immunofluorescence of cell-specific markers. Anti–von Willebrand factor (E), anti–-actin (F), anti–prolyl-4-hydroxylase (G), and anti-CD68 (H) antibodies were used as markers of ECs, SMCs, fibroblasts, and macrophages, respectively. Lower panels show double-immunofluorescence (I through L). Colocalization of cell-specific markers and GRX is shown by yellow-labeled immunofluorescence. Results are representative of 3 experiments.

Comparison of Localization of GRX With TRX in Human Coronary Arteries
Next, the localization of TRX, another member of the thiol-disulfide oxidoreductases, in human coronary arteries was examined. As shown in Figure 3, the expressional pattern of TRX was quite similar to that of GRX. In nonatherosclerotic coronary arteries, TRX was mainly expressed in medial SMCs. In atherosclerotic coronary arteries, the expression of TRX was increased through the vessel wall, especially in infiltrating macrophages.

View larger version (54K): [in this window] [in a new window]   Figure 3. Comparison of expression of GRX and TRX in human coronary arteries. Expression of GRX in nonatherosclerotic (A) and atherosclerotic (D) coronary arteries. Expression of TRX in nonatherosclerotic (B) and atherosclerotic (E) coronary arteries. The expressional pattern of TRX in coronary arteries was similar to that of GRX. When the primary antibody was replaced with a rabbit nonspecific immunoglobulin, no reactivity was detected (C and F). Results are representative of 4 independent experiments.

Effect of H₂O₂ on GRX Protein Expression in Human CASMCs
Because a recent report had shown that H₂O₂ stimulated GRX gene expression in E coli,¹⁹ we next examined the effect of H₂O₂ on the protein expression of GRX in cultured human CASMCs. Western blot analysis revealed that H₂O₂ significantly induced the expression of GRX in cultured human CASMCs. These responses were concentration dependent, peaked at 500 µmol/L, and then declined somewhat at 1000 µmol/L (Figure 4B). This induction was observed within 6 hours after stimulation, and it was sustained for at least 12 hours (Figure 4A). The induction of GRX 12 hours after stimulation was 2.5-fold.

View larger version (30K): [in this window] [in a new window]   Figure 4. Western blot analysis of GRX in cultured human CASMCs. Human CASMCs were cultured to confluence on 60-mm dishes. After incubation with H2O2, cell lysates were analyzed by immunoblotting with anti-GRX antibody as described in Methods. A, Cells were incubated with 500 µmol/L H2O2 for the indicated times (A) or with various concentrations of H2O2 for 6 hours (B). H2O2 increased protein expression of GRX. This response was concentration dependent, peaked at 500 µmol/L, and then declined somewhat at 1000 µmol/L. Results are representative of 3 independent experiments.

Correlation of ROS and GRX Expression
The investigation with cultured human CASMCs suggested a significant role for ROS in the expression of GRX. Therefore, the correlation of ROS and GRX expression in coronary arteries was investigated. The generation of ROS was examined by fluorescence microtopography with dihydroethidium. As shown in Figure 5, ROS was slightly detected in the endothelium, intima, media, and adventitia of nonatherosclerotic coronary arteries, whereas intense production of ROS was observed in atherosclerotic coronary arteries. ROS production was enhanced through the vessel wall, especially with accumulating macrophages in plaques, medial SMCs, and adventitial fibroblasts. Interestingly, the distribution of ROS-positive cells was closely related to GRX-expressing cells, suggesting that ROS were involved in the expression of this thiol-disulfide oxidoreductase.

View larger version (75K): [in this window] [in a new window]   Figure 5. In situ detection of ROS with confocal image of dihydroethidium and immunofluorescence of GRX in human coronary arteries. Upper panels (red) show generation of ROS as detected by the oxidative fluorescence microtopography method (A through C). Lower panels (green) show immunofluorescence of GRX (D through F). Left panels (A and D) are images of nonatherosclerotic coronary arteries, and middle and right panels (B, C, E, and F) are those of atherosclerotic coronary arteries. ROS were slightly detected in nonatherosclerotic coronary arteries (A), whereas intense production of ROS was observed in atherosclerotic coronary arteries (B and C). The production of ROS was closely associated with GRX. Results are representative of 7 experiments.

	Discussion

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Several lines of evidence have suggested that oxidative stress is involved in the pathogenesis of atherosclerosis through various mechanisms, such as stimulation of SMC proliferation, inactivation of endothelium-derived nitric oxide, and induction of redox-sensitive genes.^20,21 Although the mechanisms for mediating a redox balance in the vessel wall are not fully understood, it has become clear that NADH/NADPH oxidase is one of the key enzymes that acts as a source of ROS.^13,22 Against increased oxidative stress in the vasculature, antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase play an important role in scavenging ROS. In addition to these enzymes, a family of thiol-disulfide oxidoreductases works as cytoprotective antioxidants.^8,23–25 In the present study, we demonstrated that GRX and TRX were expressed in ECs, fibroblasts in the adventitia, and most intensely, SMCs in the media in human coronary arteries. Interestingly, in atherosclerotic lesions such as hypercellular lesions and fibrous plaques, the infiltrating macrophages, which are known to produce various ROS, highly expressed GRX. The expressional pattern of TRX was quite similar to that of GRX. Furthermore, in cultured CASMCs, H₂O₂ stimulated the expression of GRX in a time- and dose-dependent manner. Fluorescence microtopography with dihydroethidium demonstrated that the generation of ROS was associated with GRX expression.

The significance of the TRX/GRX system in the pathogenesis of coronary artery disease is still unknown; however, our results suggest some possibilities of the roles of the TRX/GRX system in the vessel wall. First, because TRX as well as GRX was intensely expressed in medial SMCs, even in nonatherosclerotic coronary arteries, it may have an important antioxidative effect under normal vessel conditions. Second, the induction of TRX and GRX in macrophages in atherosclerotic lesion may be a protective system against the increased production of ROS in response to the progression of atherosclerosis. The expressional changes of GRX and TRX were quite similar, suggesting that these thiol-disulfide oxidoreductases may synergistically protect vascular cells against enhanced oxidative stress in atherosclerotic lesions.

Recently, we demonstrated that the expression of p22phox, an essential component of NADH/NADPH oxidase, in human coronary arteries was increased with the progression of atherosclerosis, suggesting that the oxidative stress was likely enhanced in atherosclerotic vessels.¹³ Because the TRX/GRX system is 1 of the important factors that maintains the redox environment in the cell, induction of this system as observed in the present study may be a counterregulatory mechanism against the increased oxidative stress in atherosclerotic vessels.

In atherosclerotic coronary arteries, the macrophages infiltrating into the fibrous plaques and hypercellular lesions were strongly positive for GRX and TRX. The mechanisms for mediating regulation of the TRX/GRX system in coronary vessels remain to be elucidated; however, there are several possibilities to explain their induction in atherosclerotic plaques. Recently, Takashima et al²⁶ reported that the expression of GRX was increased during monocyte differentiation to macrophages. Therefore, the differentiation of monocytes might regulate the expression of thiol-disulfide oxidoreductases. Takagi et al¹¹ suggested that nitric oxide produced by inducible nitric oxide synthase plays a crucial role in the induction of TRX in atherosclerotic plaques. Although there are some controversies about the expression of nitric oxide synthase in atherosclerotic plaques, nitric oxide might regulate expression of the TRX/GRX system. On the other hand, in the present study, H₂O₂ increased the protein expression of GRX in cultured SMCs. Furthermore, fluorescence microtopography with dihydroethidium demonstrated that the generation of ROS was closely associated with GRX, suggesting a role for ROS in its induction. However, all ROS-generating cells did not express GRX, and the overlapping of dihydroethidum staining and GRX expression was not complete. Therefore, some other factors might be involved in its induction in atherosclerotic arteries.

Redox regulation by covalent modification of the thiol group has been considered to play a critical role in signal transduction. Recently, Saitoh et al⁷ demonstrated the association of TRX with apoptosis signal–regulating kinase (ASK1), a central kinase, in regulating apoptosis in vivo and in vitro. The interaction between TRX and ASK1 depended on the redox state of TRX and inhibited ASK1 kinase activity. Given the functional similarity of GRX and TRX, the cellular redox state modified by GRX might be involved in the pathogenesis of atherosclerotic coronary artery disease via its antioxidant effect and/or its role as a signaling molecule. Although further experiments will have to be done to understand the role of thiol-disulfide oxidoreductases in coronary artery disease, our findings provide a new insight into the pathogenesis of atherosclerotic coronary diseases.

	Acknowledgments

 
This study was supported by the Uehara Memorial Foundation and grants in aid for scientific research from the Ministry of Education, Science, and Culture (11670679, 12835007, 12877109, 12470154).

We thank Kiyoko Matsui for excellent technical assistance.

Received December 14, 2000; accepted June 25, 2001.

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