Role of oxidative stress in adaptive responses in special reference to atherogenesis.

Although lipid peroxidation products have been implicated in oxidative stress-related diseases, pretreatment of cells with such compounds at sublethal concentrations shows significant cytoprotective effects against forthcoming oxidative stress. The adaptive response induced by 4-hydroxynonenal (HNE) is critically mediated by gene expression of cytoprotective proteins via NF-E2-related factor 2/Kelch-like-ECH-associated protein 1 (Nrf2/Keap-1) pathway. The physical or mechanical stimuli such as shear stress also impose adaptive responses by inducing gene expression. Laminar shear stress, anti-atherogenic shear stress activates Nrf2/Keap-1 pathway. The transcriptome analysis using DNA microarray reveal high similarity in gene expression profiles of cells treated with HNE and laminar shear stress, providing insight into molecular mechanisms. These findings suggest a general hormetic effect of diverse stimuli in cell cultures and may lead to a reappraisal of the eventual role of reactive oxygen species and lipid peroxidation in organisms.

Introduction

The generation of reactive oxygen species (ROS) and subsequent oxidative modification of biomolecules, such as lipids, proteins, and nucleic acids, are inevitable in aerobic organisms. An excessive amount of ROS has been implicated in a variety of pathological events such as atherosclerosis, ischemia-reperfusion injury, cardiovascular diseases, and neurodegenerative diseases [1]. “Oxidative stress” was defined as a disturbance in the prooxidant-antioxidant balance in favor of the former [2]. When the stress level exceeds defense capacity, it may induce oxidative damage.

Above all, lipid peroxidation has received much attention and has been accepted to cause disturbance of fine structure and functional loss of biological membranes and to produce some toxic products. Phosphatidylcholine hydroperoxide is the primary product of lipid peroxidation, which undergoes non-enzymatic reactions, leading to the formation of 4-hydroxynonenal (HNE) and malondialdehyde, secondary products of lipid peroxidation [3, 4]. Alternatively, the lipid hydroperoxides, especially linoleic acid hydroperoxide, can be also oxidized enzymatically by some peroxygenases and reductases into hydroxyoctadecadienoic acids [5, 6]. Lysophosphatidylcholine (lysoPC) [7] is generated from phosphatidylcholine during oxidative modification of low density lipoprotein (LDL), which is known as a key event in atherogenesis [8]. Oxysterols are defined as oxygenated derivatives of cholesterol that may be formed directly by autoxidation or by the action of a specific monooxygenase [9].

It is widely accepted that all these lipid peroxidation products mentioned above could induce oxidative stress and be involved in the pathogenesis of a number of degenerative diseases [9-11]. However, recent studies have revealed that low levels of ROS and lipid oxidation products may play essential roles in the cell signal transduction [12, 13] and can induce adaptive response [14-20]. In another word, the low level stress may stimulate defense network and acts as a good stress, “eustress” [21-23]. In addition to these chemical or biochemical molecules, physical forces such as pressure and shear stress act as stress inducer (oxidative stress inducer) to cells. Since cells respond to oxidative stress via expression of genes, the transcriptome analysis using DNA microarray provides a useful data to determine a large spectrum of changes associated with stress in cells.

Global Analysis of Gene Expression using DNA Microarray

Atherosclerosis, leading to coronary heart disease and stroke is the most common cause of death in industrialized nations. It has been suggested that oxidative modification of LDL is a key initial event in the development of atherosclerosis [8]. A wide variety of oxidized lipids and oxidized LDL (oxLDL) itself have been detected in atherosclerotic lesions [24]. The esterified fatty acids of phosphatidylcholine and cholesteryl ester are oxidized enzymatically and non-enzymatically in vitro to yield lipid hydroperoxides as primary products [25], followed by secondary reactions to form lipid hydroxides and aldehydes such as malondialdehyde, acrolein [4] and HNE. Acrolein and HNE are known to be highly reactive and to form adducts with proteins and nucleic acids [26]. In particular, many studies have shown that HNE regulates cell-signaling pathways through activation protein 1 (AP-1) [27-29]. HNE is also known to act as an electrophile resulting in activation of transcription factor, NF-E2 related factor 2 (Nrf2) by releasing it from Kelch-like ECH-associated protein-1 (Keap-1) [30, 31]. Oxidized PC is more susceptible than PC itself for the reaction of phospholipase A2 to give LysoPC which is present at high concentration in oxLDL [32]. LysoPC is known to induce adhesion molecules expression in endothelial cell [33-35] and cytokines by monocytic cells [36]. Cholesterol is also oxidized to give several classes of oxysterols: 7-ketocholesterol (7-keto), which induces monocyte differentiation and promote foam cell formation [37]; 22(R)-hydroxycholesterol (22-ROH), which is a ligand for the liver X receptor and regulates the expression of genes involved in cholesterol and fatty acid homeostasis [38]; and 25-hydroxycholesterol (25-OH), which regulates cholesterol synthesis via the sterol regulatory element-binding protein (SREBP)/SREBP cleavage activating protein regulatory pathway [39].

The technology of DNA microarray provides a useful tool to determine a large spectrum of responses of cells treated with each lipid peroxidation product and oxLDL. We carried out large-scale gene expression analysis using human endothelial cell exposed to oxLDL and lipid peroxidation products such as LysoPC, HNE, 7-keto, 22-ROH and 25-OH. All results of clustering analysis are shown in Figure 1, which suggest the properties of oxLDL-regulating gene expression in endothelial cell and lipid components responsible for oxLDL-induced gene expression.

Fig. 1

Clustering analysis of gene expression induced by lipid peroxidation products. HUVEC were treated with LysoPC, HNE, 7-keto, 22-ROH, and 25-OH for 1 and 4 h, and nLDL and oxLDL for 4 and 8 h. Gene expression was analyzed by DNA microarray (GeneChip®). Up-regulated genes and down-regulated genes are shown in red color and green color, respectively.

Human umbilical vein endothelial cells (HUVEC) were treated with 200 µg/mL native LDL, 200 µg/ml oxLDL, 10 µmol/l 7-keto, 10 µmol/l 22-ROH, 10 µmol/l 25-OH, 30 µmol/l LysoPC, or 5 µmol/l HNE for 1(n = 1), 4 (n = 3), and 8 h (n = 3 only for oxLDL). Genes represented in red color and green color are up-regulated and down-regulated, respectively. The 270 genes were classified into 5 clusters basically, A: decreased by oxLDL and/or lipid peroxidation products in oxLDL; B: increased by oxLDL and lipid peroxidation products; C: increased by oxLDL and HNE; D: increased by LysoPC; E: increased by LysoPC but decreased by oxLDL and/or oxysterols.

In this review, the cluster C containing genes up-regulated by oxLDL and HNE is focused on since this cluster provides a hint leading us to an idea of adaptive response of cells. More than 40 % genes in cluster C is regulated by Nrf2 (* in Table 1). HNE acts as an electrophile with its carbonyl group and oxidizes Keap-1 to release Nrf2 resulting in expression of Nrf-2 regulating genes. Although the concentration of free HNE in oxLDL used in this study (0.5 µM), was much smaller than that of HNE added directly into medium (5 µM), oxLDL reproduced induction of all genes induced by HNE. OxLDL is capable of eliciting ROS generation and enhance lipid peroxidation [40] and the secondary oxidation products such as acrolein as well as HNE and ROS might oxidize Keap-1 to release Nrf2 [41]. HNE and oxLDL activate cyclooxygenase resulting in production of 15-deoxy-delta12,14-prostaglandin J2 which is known to enhance release of Nrf2 from Keap-1 [42].

Table 1

Up-regulated genes by HNE and oxLDL

Gene name	Full name of gene	Fold change mean +/− SD
		4HNE_(4 h)	OxLDL (8 h)	OxLDL (4 h)	LysoPC (4 h)
HMOX1	heme oxygenase (decycling) 1*	19.8 ± 10	45.32 ± 11.54	39.09 ± 8.49	2.75 ± 1.41
HSPA1A	heat shock 70 kDa protein 1A	5.93 ± 2.84	37.54 ± 22.98	23.36 ± 7.51	0.65 ± 0.07
OKL38	pregnancy-induced growth inhibitor	5.04 ± 0.2	14.19 ± 3.14	6.53 ± 0.75	1.55 ± 0.95
HSPA1B	heat shock 70 kDa protein 1B	3.98 ± 1.97	10.68 ± 2.03	10.91 ± 1.93	0.93 ± 0.06
SLC7A11	solute carrier family 7, (cationic amino acid transporter)*	3.78 ± 0.72	4.38 ± 2.02	3.57 ± 1.24	1.87 ± 0.22
GCLM	glutamate-cysteine ligase, modifier subunit*	3.55 ± 0.84	5.08 ± 0.85	4.1 ± 0.32	1.35 ± 0.05
SQSTM1	sequestosome 1*	2.98 ± 1.18	5.73 ± 1.75	3.01 ± 0.63	1.18 ± 0.05
ERBBP	estrogen-responsive B box protein	2.69 ± 0.68	7.07 ± 1.48	2.56 ± 0.52	0.99 ± 0.28
TRIM16	tripartite motif-containing 16*	2.69 ± 0.68	4.48 ± 1.1	2.56 ± 0.52	0.99 ± 0.28
GR	glutathion reductase*	2.56 ± 0.87	3.97 ± 0.31	1.72 ± 0.7	1.16 ± 0.35
TR1	thioredoxin reductase 1*	2.47 ± 0.17	2.56 ± 0.81	2.28 ± 0.48	1.49 ± 0.22
TNFSF18	tumor necrosis factor (ligand) superfamily, member 18	2.44 ± 0.51	2.25 ± 1.04	2.03 ± 0.42	1.3 ± 0.39
APG-1	heat shock protein (hsp110 family)	2.31 ± 0.32	6.68 ± 8.08	2.22 ± 0.73	0.84 ± 0.42
AKR1C3	aldo-keto reductase family 1, member C3	2 ± 0.14	3.62 ± 1.15	1.98 ± 0.42	1.12 ± 0.37
H11	protein kinase H11	1.88 ± 0.23	11.68 ± 1.95	4.56 ± 0.95	1.56 ± 0.54
NQO1	NAD(P)H dehydrogenase, quinone 1*	1.87 ± 0.13	2.36 ± 0.99	1.66 ± 0.07	1.29 ± 0.05
GTR2	Rag C protein	1.87 ± 0.54	2 ± 0.51	1.45 ± 0.11	1.16 ± 0.2
RIT1	Ras-like without CAAX 1	1.84 ± 0.31	4.11 ± 0.44	2.31 ± 0.29	1.4 ± 0.29

*: Nrf2-regulated gene

Adaptive Response induced by Lipid Peroxidation Products

HNE is one of the major end products of lipid peroxidation, and has been found to induce oxidative stress, involving in the pathogenesis of a number of degenerative diseases such as Alzheimer’s disease [43], atherosclerosis [44], cataract [45], and cancer [46]. However, increasing evidence has suggested that HNE at low concentrations takes an important role in cell signal transduction and gene expression [47-52]. HNE can react in Michael additions across its carbon-carbon double bond with a wide variety of cellular components, including DNA and proteins [4]. Thus, it has been suggested that HNE could act as a potential activator of Nrf2 and induce the expression of phase II detoxification enzymes [53-54].

We have recently found that stimulation with sublethal concentrations of HNE can induce adaptive response and protect neural PC12 cells against the subsequent oxidative stress [21, 22]. There are mainly two cytoprotective systems against oxidative stress named glutathione system and thioredoxin system in cell (Fig. 2). Interestingly, both glutathione reductase (GR) and thioredoxin reductase (TR) which are a member of glutathione system and thioredoxin system, respectively, are listed in Table 1. We then attempted to seek for the underlying molecular mechanisms responsible for such an adaptive response in PC12 cells. The cellular antioxidative glutathione system, including cellular glutathione peroxidase, glutathione s-transferase, GR and total glutathione contents, did not show any considerable changes in cells treated with HNE for 24 h. In contrast, mRNA levels and activity of thioredoxin reductase-1 (TR1) were significantly elevated by the treatment with sublethal concentrations of HNE [21]. Furthermore, critical roles of TR1 in adaptive response in PC12 cells were confirmed by siRNA transfection. In fact, the data provided by DNA microarray analysis of gene expression induced by HNE in HUVEC encouraged us to investigate roles of TR1 in PC12 (Table 1).

Fig. 2

Protective systems of cell against oxidative stress. There are two main cytoprotective systems against oxidative stress, called “glutathione system” and “thioredoxin system”.

Sensing of Oxidative Stress induced by Shear Stress

Cells respond to physical and mechanical stimuli as well as chemical stimuli. Vascular endothelial cells are constantly subjected to the shear stress imposed upon them by blood flow. Atherosclerotic lesions are likely to develop focally at bifurcations and branch points in the vessel [55, 56]. It has been reported that the most vulnerable regions are exposed to non-unidirectional, disturbed or oscillatory flow and that atherosclerosis resistant regions are exposed to unidirectional laminar flow [55, 57]. To investigate the response of endothelial cells upon exposure to shear stress, many studies have been performed under a variety of experimental conditions with various flow-exposing apparatus. Many of these studies have focused on the expression of vascular cell adhesion molecule-1 (VCAM-1), since VCAM-1 has been accepted as a good marker for the endothelial phenotype in atherosclerosis-prone regions [58-61]. Endothelial cells express VCAM-1 upon exposure to non-unidirectional flow, that is, oscillatory flow at a low shear stress (+/− 5 dyn/cm2, mean time-averaged shear stress of 0.2 dyn/cm2) [62]. In contrast, endothelial cells exposure to unidirectional steady flow at high shear stress (>10 dyn/cm2) do not express VCAM-1 [63] or even suppress it [61].

To explore candidates responsible for anti-atherogenicity of laminar shear stress, we performed DNA microarray analysis after exposure of HUVEC to laminar shear stress (mean shear stress <0.2 dyn/cm2). In response to laminar shear stress, Nrf-2- dependent genes such as heme oxygenase-1 (HO-1), sequestsome 1 (SQSTM1), solute carrier family number 7 A 11 (SLC7A11) (or xCT), TR1, glutamate cysteine ligase modifier (GCLM), NAD(P)H dehydrogenase, quinone-1 (NQO-1), and Tripartite motif-containing 16 (TRIM16) were significantly induced (* in Table 2) [64]. The high similarity of gene expression induced by laminar shear stress to that induced by HNE give a rise of a hypothesis in which electrophiles such as HNE might be generated in cells upon exposure to shear stress (Fig. 3). Further studies suggest that shear stress stabilizes Nrf2 protein via the lipid peroxidation elicited by xanthine oxidase and flavoprotein (NADPH oxidase, etc.) mediated generation of superoxide, resulting in lipid peroxidation and gene induction by the Nrf2-ARE (antioxidant response element) signaling pathway [65].

Table 2

Up-regulated genes by laminarr shear stress

Gene name	Full name of gene	static	laminar shear stress
		average difference	average difference	fold change
HMOX1	heme oxygenase (decycling) 1*	277	4904	18.0
SQSTM1	sequestosome 1*	42	411	7.2
HSPA1A	heat shock 70 kDa protein 1A	130	962	5.7
SLC7A11	solute carrier family 7A11*	397	1947	4.9
TRIM16	tripartite motif-containing 16*	71	320	4.7
PMCH	Pro-melanin-concentrating hormone	8	235	4.2
SLC3A2	solute carrier family 3A2	97	443	4.2
TR1	thioredoxin reductase 1*	727	2773	4.0
GCLM	glutamate-cysteine ligase, modifier subunit*	152	607	4.0
EEF1A1	eukaryotic translation elongation factor 1 alpha 1	2931	10384	3.8
NQO1	NAD(P)H dehydrogenase, quinone 1*	516	2263	3.8
PTGS2	prostaglandin-endoperoxide synthase 2	38	202	3.8

*: Nrf2-regulated gene

Fig. 3

Roles of lipid peroxidation products in cytoprotective response of cell upon exposure to laminar shear stress. ROS generation occurs in response to shear stress and induces lipid peroxidation. The expression of genes encoding cytoprotective proteins are induced by Nrf2/ARE signaling pathway which are activated by lipid peroxidation products.