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(Circulation. 1998;97:1071-1078.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Induction of Rat Aortic Smooth Muscle Cell Growth by the Lipid Peroxidation Product 4-Hydroxy-2-Nonenal

Johannes Ruef, MD; Gadiparthi N. Rao, PhD; Fengzhi Li, PhD; Christoph Bode, MD; Cam Patterson, MD; Aruni Bhatnagar, PhD; ; Marschall S. Runge, MD, PhD

From the Division of Cardiology and Sealy Center for Molecular Cardiology, the Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch at Galveston; and the Division of Cardiology (C.B.), University of Heidelberg (Germany).

Correspondence to Marschall S. Runge, MD, PhD, University of Texas Medical Branch, Division of Cardiology, 5.106 John Sealy Hospital, 301 University Blvd, Galveston, TX 77555-0553. E-mail mrunge{at}utmb.edu

	Abstract

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Background—Atherosclerotic lesion formation is a complex process, in part mediated by inflammatory and oxidative mechanisms including lipid peroxidation. To further characterize the potential role of lipid peroxidation products in atherogenesis, we studied the effects of 4-hydroxy-2-nonenal (HNE) on rat aortic smooth muscle cell growth.

Methods and Results—HNE, at concentrations of 1.0 and 2.5 µmol/L, significantly stimulated rat aortic smooth muscle cell growth as determined by cell counts, [³H]-thymidine uptake, and incorporation of bromo-deoxyuridine. To characterize the mechanism of HNE-induced mitogenesis, its effect on activation of intracellular growth signaling pathways was examined. Treatment with HNE resulted in activation of extracellular signal-regulated protein kinases ERK1 and ERK2, induction of c-fos and c-jun protein expression, and an increase in transcription factor AP-1 DNA binding activity. In addition, HNE induced expression of platelet-derived growth factor-AA (PDGF-AA) protein, and an anti–PDGF-AA antibody specifically inhibited HNE-mediated DNA synthesis, suggesting that growth factor induction may play a role in HNE-induced vascular smooth muscle cell growth. The role of redox-sensitive mechanisms in this process was further supported by the observation that HNE-induced DNA synthesis and AP-1 activation were inhibited by the antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate.

Conclusions—These data demonstrate that HNE, one of several important lipid peroxidation products, induces rat aortic smooth muscle cell growth through redox-sensitive mechanisms and growth factor expression. These observations are consistent with a role for lipid peroxidation products in vascular smooth muscle cell growth in atherogenesis.

Key Words: atherosclerosis • oxidation • lipids • signal transduction • mitogens

	Introduction

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Atherosclerosis is a multifactorial disease that ultimately results in thickening of the arterial wall with a concomitant decrease in lumen diameter. Two mutually compatible theories have been proposed to explain the development of atherosclerotic lesions. The "oxidation" hypothesis of atherogenesis emphasizes oxidative modification of lipoproteins leading to recruitment of macrophages and other inflammatory cells into lesions as the key event in atherogenesis.^{1 2} The "response-to-injury" hypothesis posits VSMC proliferation as a hallmark of the formation and progression of atherosclerotic lesions.³

Although a large body of data supports a role for both oxidative modification of lipids and VSMC mitogenesis in atherogenesis, mechanistic links between these two events have only recently been proposed. After an insult to the vessel wall, inflammatory mechanisms of protection and repair are initiated, an important component of which is the generation of reactive oxygen species (ROS). Activated leukocytes produce superoxide anion, hydrogen peroxide, and hydroxyl radicals through activation of NADH/NADPH oxidases.^{4 5} In addition, endothelial cells and VSMC are a source of significant free radical production.⁶ These locally generated ROS result in oxidation of both circulating and membrane-bound lipids, perhaps accounting for the increased levels of lipid peroxidation products reported in patients with atherosclerosis.⁷ It has also been demonstrated that ROS and oxidized lipids both stimulate VSMC growth.^{8 9 10 11 12} Furthermore, PDGF, which has been implicated in VSMC proliferation in atherosclerosis, requires ROS generation for its mitogenic effects.¹³ Finally, antioxidants such as probucol, ß-carotene and -tocopherol inhibit vascular lesion formation in hyperlipidemic animals^{14 15 16} and thiol antioxidants, such as PDTC, inhibit vascular cell growth in vitro.¹⁷ Together, these data support a mechanistic relationship between oxidant generation, lipid peroxidation, VSMC growth, and atherogenesis.^{18 19}

In the present study, we hypothesized that HNE, a component of oxidatively modified lipids, might link oxidative events to VSMC proliferation in atherogenesis. HNE is a major product of lipid peroxidation that is produced by ß-scission of alkoxyl radicals in polyunsaturated fatty acids such as arachidonic, linoleic, and linolenic acids that are present in LDL particles.²⁰ HNE is detectable in the plasma of healthy probands (up to 1.4 µmol/L) and is present in tissues at concentrations of up to 20 µmol/L.^{21 22} Several observations support the hypothesis that HNE may provide a link between oxidant generation, lipid peroxidation, and VSMC proliferation in atherogenesis. First, HNE is a component of oxidized LDL and is found in atherosclerotic lesions.²³ Immunoreactive HNE is present at all stages of human atherosclerosis but not in normal human arteries²⁴ and has also been identified in the neointima of balloon-injured baboon arteries.²⁵ Second, lipid peroxidation products, and specifically HNE, stimulate chemotaxis and growth in other systems^{26 27 28} and have been implicated in other pathological conditions thought to be related to oxidative stress.^{29 30 31} In these settings, it has been proposed that HNE and related aldehydes act to amplify the cellular effects of their free radical precursors.²¹

Here we report that HNE stimulates proliferation of RASM. This dose-dependent proliferation is associated with induction of mitogenic signaling events in RASM, including activation of ERKs, increases in c-fos and c-jun protein expression, and enhanced AP-1-DNA binding activity. HNE induces PDGF-AA protein expression and an anti-PDGF-AA antibody ameliorates the effects of HNE on RASM growth, suggesting that growth factor induction may be an important intermediary step in HNE-induced vascular smooth muscle cell growth. Finally, the effects of HNE on proliferation and mitogenic signaling are inhibited by the antioxidants NAC and PDTC, and HNE rapidly downregulates cellular thiol groups, suggesting that the effects of HNE are at least partly mediated through redox-sensitive events.

	Methods

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Materials
Unless otherwise mentioned, all chemicals were purchased from Sigma Chemical Co. HNE was obtained from Cayman Chemical Co. [methyl-³H]-thymidine was obtained from DuPont NEN and [-³²P]ATP from Amersham Co. Antibodies for c-fos, c-jun, PDGF-AA, FLK-1, and rabbit IgG (PE) were from Santa Cruz Biotechnology Inc, Oncogene Science, Genzyme, and Boehringer Mannheim, respectively. Double-stranded oligonucleotides containing a consensus AP-1 recognition sequence were purchased from Promega.

Cell Culture
RASM were isolated from the thoracic aortas of 200- to 250-g male Sprague-Dawley rats by enzymatic digestion and kept in culture as described previously.^{25 32} Three different RASM isolates were used. For most experiments, cells at 80% confluency were made quiescent by incubation for 72 hours in DME containing 0.1% fetal bovine serum. For cell number and DNA synthesis experiments, cells were grown to 50% confluency. RASM were used at passages 6 to 15, since no differences in responsiveness were noted within this range. Low levels of serum were maintained during quiescence to prevent slow apoptosis that accompanies complete serum deprivation in vascular smooth muscle cells.^{33 34}

Cell Number
Cells were growth-arrested in 60-mm dishes, as described above, and HNE or serum was added to the cells. After 72 hours the cells were trypsinized, washed in PBS, and counted in a Coulter counter (Coulter Electronics).

DNA Synthesis
Growth-arrested RASM were treated with HNE or 10% serum for 48 hours. [³H]-thymidine (1 µCi/mL) was added 24 hours before the end of the incubation period, and DNA synthesis was measured as trichloroacetic acid-precipitable material as described previously.²⁵ The experiments including neutralizing antibodies (0.2 µg/mL) were performed with RASM growth-arrested in serum-free (0%) medium.

Cell Proliferation ELISA
An ELISA kit (Biotrak, Amersham), based on the incorporation of BrdU, was used and the assay was performed following the manufacturer's guidelines. In brief, RASM were growth-arrested in 96-well plates and treated with serum or HNE in the presence of BrdU for 24 hours. After fixation and blocking, a peroxidase-labeled anti-BrdU antibody was added. The substrate reaction was performed with tetramethylbenzidine, and the color was read at 450 nm in a spectrophotometer (Molecular Devices).

Western Blot Analysis
After treatment of growth-arrested RASM with the indicated agents, cell lysates were prepared and immunoblotting was performed as described previously.²⁵

In-Gel Kinase Assay
Equal amounts of protein (50 µg/lane) were resolved by 0.1% SDS–10% PAGE.³⁵ The gel was copolymerized with myelin basic protein, treated with [-³²P]ATP, and exposed to autoradiography as described previously.³⁶

Electrophoretic Mobility Shift Assay
Growth-arrested RASM were treated for various time periods with and without the indicated agents. Nuclear extracts were prepared according to methods previously described.³⁷ Protein-DNA complexes were formed using 5 µg of nuclear protein and 100,000 cpm of [³²P]-labeled AP-1 oligonucleotide probe (5'-CGCTTGATGAGTCAGCCGGAA-3'). Protein-DNA complexes were resolved on a 5% polyacrylamide gel, as described previously.¹²

Thiol Group Assay
After treatments of growth-arrested RASM with 1.0 µmol/L HNE, 10% serum, or BSO, 1 mmol/L, an inhibitor of glutathione synthase³⁸ for appropriate times, cells were washed twice with PBS and lysed by repeated freeze-thawing. Thiol groups were determined using Ellman's reagent as described. Light absorbance at 412 nm was used to calculate cellular thiol groups with E₄₁₂=1.4x10⁴xmol/L^-1xcm^-1.³⁹

Data Analysis
Data are expressed as mean±SEM. For multiple treatment groups, one-way ANOVA followed by Bonferroni's t test was applied. Values of P<.05 were considered significant.

	Results

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HNE Induces RASM Growth
To determine whether HNE stimulates vascular smooth muscle cell growth, we examined the effect of HNE on RASM cell number and DNA synthesis. After exposure of growth-arrested RASM to HNE (1.0 to 5.0 µmol/L), cell number was determined after 3 days of treatment. Cell number was significantly increased after treatment with either 1.0 µmol/L or 2.5 µmol/L HNE (by 41% and 37%, respectively, P<.05) compared with controls (Fig 1A). These effects of HNE on cell number were similar in magnitude to the mitogenic effects of other growth factors that have been studied⁴⁰ but less than that of 10% serum. Interestingly, at a higher concentration (5.0 µmol/L), HNE treatment resulted in a decrease in cell number by 34% compared with control (P<.05). This may have been due to the known toxicity of HNE when used at this concentration for extended time periods.⁴¹ HNE treatment of RASM induced a similar effect on DNA synthesis as determined by measurement of [³H]-thymidine incorporation. Treatment with HNE at 1.0 µmol/L and 2.5 µmol/L over 48 hours increased [³H]-thymidine incorporation by 41% and 30%, respectively (P<.05, Fig 1B). As with cell growth, treatment of RASM with a higher dose of HNE (5.0 µmol/L) resulted in decreased DNA synthesis. To confirm these results, we also measured the effect of HNE on DNA synthesis by determining incorporation of BrdU in RASM, both in vitro (Fig 1C) and ex vivo in cultured rat aorta sections (data not shown). As shown in Fig 1C, treatment of RASM with HNE at 1.0 µmol/L and 2.5 µmol/L over 24 hours resulted in increased incorporation of BrdU (110% and 82%, respectively, P<.05) compared with controls. Additional experiments were performed to determine whether pulse-labeling with [³H]-thymidine and quiescence of RASM in 0% serum would maximize the mitogenic effect of HNE. Although a slightly more marked effect was observed under these conditions (see Fig 6), prolonged lack of serum can lead to apoptosis in RASM and other VSMC,^{33 34} potentially confounding the effects of higher doses of HNE. For this reason, the majority of the experiments reported here were performed using the standard conditions described in the "Methods" section. Together these data indicate that HNE induces RASM growth.

View larger version (15K): [in this window] [in a new window]   Figure 1. HNE increases RASM number and DNA synthesis. A, Growth-arrested RASM were treated with and without HNE in the doses indicated or 10% serum for 3 days and cells were counted using a Coulter counter. Results are shown as mean±SEM (n=9). B, Growth-arrested RASM were treated with and without HNE or serum in the doses indicated in the presence or absence of NAC (20 mmol/L) for 48 hours. Cells were exposed to 1 µCi/mL [3H]-thymidine for the last 24 hours in the 48 hour incubation period and trichloroacetic acid-precipitable material was measured as a marker of DNA synthesis. Results are shown as mean±SEM (n=6). C, To obtain additional quantification of DNA synthesis, a sensitive immunoassay based on BrdU incorporation was performed. Growth-arrested RASM were treated with or without HNE or serum for 24 hours in the presence of 10 µmol/L BrdU. Incorporation of BrdU was analyzed by ELISA. Results are expressed as mean±SEM of 9 replicates. *P<.05 compared with control.

View larger version (16K): [in this window] [in a new window]   Figure 6. HNE-induced DNA synthesis is mediated by growth factors. Growth-arrested RASM (0% serum) were treated with HNE (1 µmol/L) in the presence or absence of a neutralizing polyclonal PDGF-AA antibody (PDGF-AA Ab) or a polyclonal FLK-1 antibody (FLK-1 Ab). DNA-synthesis was measured as in Fig 2, except that a pulse-labeling for 4 hours was performed with 1 µCi/mL [3H]-thymidine. Results are shown as mean±SEM (n=3). *P<.05 compared with control; +P<.05 compared with treatment with HNE.

HNE Activates Early Mitogenic Signaling Events in RASM
The ERKs family of mitogen-activated protein kinases is a major pathway by which information from extracellular signaling events is transduced to the nucleus. Because ERKs are implicated in mitogenic responses in VSMC induced by ROS such as H₂O₂ and O₂^-,^{36 42} we examined whether HNE also activates the ERKs signaling pathway. ERKs activities were measured in HNE-treated and untreated RASM with an in-gel kinase assay, with myelin basic protein as a substrate. In this assay, after fractionation of cell lysates by SDS-PAGE and renaturation, ERKs activity is determined based on phosphorylation of myelin basic protein contained in the gel. HNE (2.5 µmol/L) rapidly and transiently activated both ERK1 and ERK2 (Fig 2). Maximal activation of both ERK1 and ERK2 (fourfold as determined by densitometry) occurred within 5 minutes. By 40 minutes, the activities of these enzymes had returned to basal levels. This time course is similar to that shown in VSMC for O₂^- and angiotensin II and more rapid than that for hydrogen peroxide.^{36 42 43} These data indicate that at least one effect of HNE is to rapidly activate critical mitogenic signaling pathways in RASM.

View larger version (21K): [in this window] [in a new window]   Figure 2. HNE stimulates extracellular signal-regulated protein kinases group of mitogen-activated kinases. Growth-arrested RASM were treated with and without 2.5 µmol/L HNE for the indicated times and cell lysates were prepared; 50 µg protein was resolved on 0.1% SDS–10% polyacrylamide gels that were copolymerized with myelin basic protein. ERKs activities were determined with in-gel kinase assay by incorporation of [-32P]ATP into myelin basic protein. This result was reproduced in two independent experiments.

HNE Induces Mitogenic Nuclear Events in RASM
The observation that HNE activates ERK1 and ERK2 raised the possibility that the mitogenic effect of HNE is ultimately mediated by activation of the nuclear factor AP-1. AP-1 activation is a well-defined mechanism by which numerous mitogens that signal through ERK1 and ERK2 stimulate VSMC growth.^{8 44} AP-1 activation has also been implicated in ROS-mediated gene regulation.⁴⁵ To address this hypothesis, we first determined by Western blot analysis whether HNE stimulates c-jun and c-fos protein expression, because new protein expression could promote AP-1 activation. Two and 4 hours after treatment with HNE (2.5 µmol/L), c-jun and c-fos proteins were fourfold greater in treated than in untreated control cells (Fig 3A). Analogous to its effects on RASM growth, HNE stimulated c-fos and c-jun expression at low concentrations, but at higher concentrations decreased expression (Fig 3B).

View larger version (30K): [in this window] [in a new window]   Figure 3. HNE induces the expression of c-fos and c-jun. A, Growth-arrested RASM were treated with and without 2.5 µmol/L HNE for the indicated times and cell lysates were prepared. Equal amounts of total protein (20 µg) from control and treated cells were resolved on 0.1% SDS–10% polyacrylamide gels and analyzed by immunoblotting for c-fos and c-jun proteins, using the respective polyclonal antibodies. B, RASM were treated with the indicated concentrations of HNE for 1 hour, and c-fos and c-jun levels were analyzed as described above.

To determine whether these HNE-induced increases in c-fos and c-jun protein translated into increased AP-1-DNA binding activity in RASM, we performed electrophoretic mobility shift analysis, using nuclear extracts from cells either treated or not treated with HNE and a ³² P-labeled AP-1 consensus oligonucleotide. Nuclear proteins were isolated from growth-arrested RASM after treatment with HNE (Fig 4). HNE increased AP-1-DNA binding activity in a time-dependent manner, with maximum binding activity at 4 hours.

View larger version (66K): [in this window] [in a new window]   Figure 4. HNE induces AP-1 activity. Growth-arrested RASM were treated with and without 1.0 or 2.5 µmol/L HNE or 10% serum for 2 to 6 hours. Nuclear extracts (5 µg) were incubated with consensus [32P]-labeled AP-1 oligonucleotide. The AP-1–DNA complex was separated from free probe on a 5% polyacrylamide gel. Specificity of the AP-1–DNA complex was determined by addition of nonradioactive AP-1 oligonucleotide (100-fold excess) to the nuclear extract of HNE-treated cells (1 µmol/L, 2 hours) before incubation with [32P]-labeled AP-1 oligonucleotide. This result is representative of three independent experiments.

The Mitogenic Effect of HNE Is at Least Partly Mediated by PDGF
In addition to directly activating mitogenic signaling pathways, ROS may induce cell growth by stimulating the production and/or secretion of growth factors. In this setting, the growth factor could potentially amplify the mitogenic effect of ROS through an autocrine mechanism. PDGF-AA has been shown to mediate such autocrine events in RASM in response to xanthine/xanthine oxidase metabolism.⁴⁶ To determine whether similar events occur in response to HNE, cell lysates of HNE-treated RASM were immunoblotted with an antibody specific for PDGF-AA. As shown in Fig 5, treatment with HNE (0.1 to 1.0 µmol/L) for 3 hours resulted in increased PDGF-AA levels. This effect was inhibited by NAC, suggesting that HNE-induced PDGF-AA synthesis is oxidant-mediated. To determine the importance of HNE-induced PDGF-AA synthesis for HNE-stimulated RASM mitogenesis, DNA synthesis was measured in the presence or absence of an anti–PDGF-AA antibody (PDGF-AA Ab). Inhibition of PDGF-AA with PDGF-AA Ab partially blocked the increase of DNA synthesis induced by HNE (60% inhibition, P<.05, Fig 6). An unrelated antibody specific for FLK-1 (FLK-1 Ab) did not inhibit HNE-induced DNA synthesis. One explanation for these results is that PDGF-AA, through an autocrine mechanism, is partially responsible for the mitogenic effect of HNE.

View larger version (26K): [in this window] [in a new window]   Figure 5. HNE induces expression of PDGF-AA. Growth-arrested RASM were treated with or without HNE (0.1 to 1.0 µmol/L) and NAC (20 mmol/L) for 3 hours. Cellular proteins (20 µg) were resolved on 0.1% SDS–10% polyacrylamide gels and analyzed by immunoblotting, using a polyclonal antibody against PDGF-AA. Recombinant PDGF-AA protein served as a positive gel marker.

HNE-Induced Mitogenesis Involves a Redox-Sensitive Mechanism
The observation that the effect of HNE on thymidine uptake in RASM was blocked by treatment with the thiol antioxidant NAC (20 mmol/L), suggested that the effects of HNE might be mediated through redox-sensitive mechanisms (Fig 1B). To further investigate this possibility, we examined the effect of two different antioxidants on AP-1 activation by electrophoretic mobility shift assay. Both NAC (20 mmol/L) and PDTC (100 µmol/L) inhibited the HNE-induced increase in AP-1-DNA binding activity (Fig 7). Diamide was used as a positive control because it is known to induce AP-1 activation.⁴⁷ Similar inhibitory effects of NAC on HNE-induced c-fos and c-jun protein expression were also observed (data not shown). On the basis of these results, it is likely that the effects of HNE on mitogenic signaling in RASM are at least in part due to redox-sensitive mechanisms.

View larger version (68K): [in this window] [in a new window]   Figure 7. Oxidants stimulate and antioxidants inhibit AP-1 activity in RASM. Growth-arrested RASM were treated with and without indicated oxidants in the presence and absence of NAC (20 mmol/L) and PDTC (100 µmol/L) for 1 hour. Nuclear proteins were isolated and AP-1 activity was measured by electrophoretic mobility shift analysis as described in Fig 4.

To determine whether this potential redox-sensitive mechanism was dependent on reduction or oxidation of cellular thiols, we also characterized the effects of HNE on total cellular thiol content. Thiol content in HNE-treated and untreated RASM was determined using Ellman's reagent. For comparison, thiol content was also measured in RASM treated with BSO, a glutathione synthase inhibitor that depletes cellular glutathione (Fig 8).³⁸ Treatment of quiesced RASM with 1.0 µmol/L HNE for 60 minutes resulted in a 52% decrease in the cellular thiol content in comparison to untreated cells (P<.05). This effect is rapid, reaching a 26% reduction of thiol groups within 5 minutes and a 43% reduction within 15 minutes of treatment with HNE, compared with the controls (P<.05). A very similar effect was observed after treatment of RASM with 1 mmol/L BSO for 60 minutes (54% decrease in cellular thiols, P<.05). Thus HNE-mediated AP-1 activation appears to use a redox-sensitive mechanism while resulting in a decrease in total cellular thiol content. While these findings are consistent with the hypothesis that the decrease in thiol content is responsible for the effect of HNE on AP-1 activation, further investigation will be required to establish the precise molecular mechanisms involved.

View larger version (17K): [in this window] [in a new window]   Figure 8. HNE treatment decreases cellular thiol groups. Growth-arrested RASM were treated with or without HNE or BSO for the indicated times. Freeze-thawed cells were dissolved in Ellman's reagent (0.5 mmol/L) and centrifuged at 6000 rpm for 10 minutes. Total cellular thiol groups were measured at 412 nm with a spectrophotometer (E412=1.4x104xmol/L-1xcm-1). The data indicate % changes in thiol levels compared with the controls and represent mean±SEM of three replicates. *P<.05 compared with controls.

	Discussion

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In the present study we have shown that HNE induces RASM proliferation in a dose-dependent fashion and in physiological concentrations.^{21 22} This effect is associated with rapid activation of a well-defined growth-regulating intracellular signaling pathway. We demonstrated that HNE treatment activates ERKs, induces expression of c-fos and c-jun protein, and increases AP-1-DNA binding activity. We also demonstrated that HNE stimulates PDGF-AA expression and that this expression may account for sustained effects of HNE on RASM proliferation. This is of interest because PDGF-AA, like other members of the PDGF family, has been previously shown to stimulate VSMC mitogenesis through autocrine production.^{46 48 49} Finally, we show that effects on growth and signal transduction are mediated, at least in part, through redox-sensitive events.

In this study, we demonstrate that HNE has a significant effect on key intracellular signaling events thought to be important in mitogenesis. The mitogenic sequelae of HNE support the hypothesis that metastable metabolites such as HNE function as mediators for the effects of oxygen-derived free radicals.⁵⁰ The rapid and transient activation of ERKs by HNE (Fig 2) with subsequent AP-1 activation (Fig 4) is similar to results obtained with linoleic acid or H₂O₂, both modulators of growth in VSMC.^{8 11 51} Thus, these findings suggest a potentially important link between the "response to injury" and "oxidative modification" hypotheses for atherogenesis. A hallmark of the response to injury hypothesis is vascular cell growth, and lipid peroxidation is a central feature in oxidative modification of lipids. Numerous observations support the notion that HNE may provide a link between these two hypotheses. Oxidized LDL is a well-described VSMC mitogen that is intimately linked to atherogenesis.^{52 53} HNE is an active constituent of oxidized LDL, and antibodies raised against HNE-LDL stain histological samples of atherosclerotic lesions from animals and patients.⁵⁴ As with HNE, at least some of the growth modulating effects of oxidized LDL are thought to be mediated by the release of growth factors.^{55 56} Thus it is possible that the mitogenic effect of HNE described in this report may, in part, explain the growth promoting effects of oxidized LDL reported by other investigators.

Lipid peroxidative reactions take place under physiological conditions in a variety of cell types, particularly in cells that are not rapidly proliferating.⁵⁷ Of the numerous intermediates generated during lipid peroxidation, unsaturated aldehydes, such as HNE may be particularly important in view of the high concentrations in which they are generated and their propensity to react with most cellular constituents.²⁰ HNE accounts for up to 95% of the unsaturated aldehydes generated during peroxidation of 6-polyunsaturated fatty acids and has been shown to be highly reactive (Fig 9A).⁵⁸ HNE can participate in a wide array of cellular reactions ranging from redox reactions and protein modification to DNA adduct formation. Because of the , ß-unsaturated structure, HNE reacts avidly with cellular nucleophiles such as thiols (Fig 9B). The thiolate anions of glutathione and cysteine add spontaneously to the electron deficient C-3 of HNE (Michael adducts).²⁰ Similar Michael adducts are also formed with histidine and lysine. The formation of such adducts has been suggested to be the underlying mechanism by which HNE, generated in oxidized LDL, modifies the histidine and lysine residues of apolipoprotein B.^{59 60} In addition to reacting with thiols, HNE also forms Schiff's bases with cellular amines via the functional aldehyde group.

View larger version (13K): [in this window] [in a new window]   Figure 9. Chemistry of HNE. A, Formation of aldehydes during lipid peroxidation. -6-polyunsaturated fatty acids (-6-PUFA) are converted into alkoxyl radicals by peroxidation reactions. ß-Cleavage of such lipid alkoxyl radicals results in formation of unsaturated aldehydes. B, Chemical structure of HNE. Three functional groups (hydroxyl group, aldehyde group, CC double bond) are present in this molecule. Thiols such as glutathione bind to HNE through thioether linkage at carbon atom 3 (Michael addition). Through the aldehyde group HNE is able to form stable Schiff's bases with proteins.

Two independent lines of evidence implicate redox-sensitive mechanisms in the downstream growth signals activated by HNE. The first is the observation that NAC and PDTC inhibit HNE-induced mitogenic signals. Second, we demonstrated that HNE rapidly oxidizes free cellular thiol groups (Fig 8), lowering the redox state of the cell and regulating nuclear events such as activation of ERKs, AP-1 and the redox-sensitive transcription factor NF-B (J. Ruef and M.S. Runge, unpublished data). This observation merits further discussion. Two other groups have reported that the maximal effects of HNE on cellular thiols⁶¹ and reaction with glutathione⁶² occur within 60 minutes, consistent with our results. Together with our data, these findings are consistent with a relationship between HNE-induced changes in intracellular thiol levels and early signaling events. Multiple reactions may be involved in this process, as it has been reported that HNE-induced depletion of protein thiols occurs through thioether binding⁶¹ and that the reaction products, saturated aldehydes, may also undergo further rearrangements.²¹ This relates to activation of growth-related signaling in that redox-sensitive activation has been demonstrated for both the ERKs and also AP-1. Our observation that lowering intracellular thiol levels leads to activation of both ERKs and AP-1-DNA binding is also not unique. Similar findings have been reported in cells treated with BSO or diamide^{47 63 64} and in VSMC treated with oxidants.¹² Thus, oxidants such as HNE may exert direct mitogenic effects on VSMC by activating ERKs and nuclear factors such as AP-1, the activity of which may depend on the cellular redox state.⁶⁵

Consistent with previous observations, HNE was found to be toxic to cells at high concentrations while lower concentrations induced cell growth and DNA synthesis (Fig 1).^{28 66 67 68} Similar effects have been observed with other oxidation products, such as H₂O₂ and oxidized LDL, which over a narrow concentration range can cause both proliferative and cytotoxic effects.^{9 51 69} We did not address the mechanisms by which this cytoxicity occurred. It might represent aldehydic modification of key proteins and/or DNA necessary for cell viability. Equally plausible, however, is the idea that HNE may exhibit a narrow concentration range, stimulating cell growth at low concentrations and having cytotoxic effects at slightly higher concentrations. This may be relevant in atherogenesis, where cell growth, apoptosis, and necrosis are thought to contribute mutually to lesion formation.⁷⁰ At 25 and 50 µmol/L HNE caused apoptosis in VSMC to an extent comparable with the effects of 50 ng/mL TNF- (J. Ruef and M.S. Runge, unpublished data).

In summary, the data presented here are consistent with the hypothesis that oxidative stress in vascular cells establishes an amplifying circuit within which long-lived oxidants such as HNE may play a part.⁴⁹ In such a circuit, ROS generate HNE, which results in further redox-sensitive events, leading to the expression of effector molecules such as PDGF, which create even more oxidative species as a part of their own signaling program. Such a circuit may explain how transient events such as activation of mitogen-activated protein kinases lead to lasting changes in cellular behavior. In addition, cellular behavior could be modified in such a circuit by interrupting the generation of ROS at multiple points in the cascade. By virtue of their long-lived nature in comparison to intracellular oxidant sources such as hydrogen peroxide, superoxide, and hydroxyl ion, modified intermediates such as HNE may be attractive sites for intervention in such circuits.

	Selected Abbreviations and Acronyms

 

BrdU = bromo-deoxyuridine BSO = L-buthionine sulfoximine ERKs = extracellular signal-regulated protein kinase HNE = 4-hydroxy-2-nonenal NAC = N-acetylcysteine PDGF = platelet-derived growth factor PDTC = prrolidine dithiocarbamate RASM = rat aortic smooth muscle cell(s) VSMC = vascular smooth muscle cell(s)

	Acknowledgments

 
This work was supported in part by the National Heart, Lung, and Blood Institute grants HL-48667 (to M.S.R.) and HL-55477 (to A.B.), by a Grant-in-Aid from the American Heart Association (to G.N.R.), and by the scholarship Ru 620/1 to 1 from the German Research Foundation DFG (to J.R.). The authors are grateful to Joann Aaron for editorial assistance.

Received July 25, 1997; revision received October 10, 1997; accepted October 29, 1997.

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