Identification of biochemical pathways for the metabolism of oxidized low-density lipoprotein derived aldehyde-4-hydroxy trans-2-nonenal in vascular smooth muscle cells

doi:10.1016/S0021-9150(01)00454-3

Atherosclerosis

Volume 158, Issue 2, October 2001, Pages 339-350

https://doi.org/10.1016/S0021-9150(01)00454-3 Get rights and content

Abstract

Oxidation of low-density lipoproteins (LDL) generates high concentrations of unsaturated aldehydes, such as 4-hydroxy trans-2-nonenal (HNE). These aldehydes are mitogenic to vascular smooth muscle cells and sustain a vascular inflammation. Nevertheless, the processes that mediate and regulate the vascular metabolism of these aldehydes have not been examined. In this communication, we report the identification of the major metabolic pathways and products of [³H]-HNE in rat aortic smooth muscle cells in culture. High-performance liquid chromatography separation of the radioactivity recovered from these cells revealed that a large (60–65%) proportion of the metabolism was linked to glutathione (GSH). Electrospray mass spectrometry showed that glutathionyl-1,4 dihydroxynonene (GS-DHN) was the major metabolite of HNE in these cells. The formation of GS-DHN appears to be due aldose reductase (AR)-catalyzed reduction of glutathionyl 4-hydroxynonanal (GS-HNE), since inhibitors of AR (tolrestat or sorbinil) prevented GS-DHN formation, and increased the fraction of the glutathione conjugate remaining as GS-HNE. Gas chromatography–chemical ionization mass spectroscopy of the metabolites identified a subsidiary route of HNE metabolism leading to the formation of 4-hydroxynonanoic acid (HNA). Oxidation to HNA accounted for 25–30% of HNE metabolism. The formation of HNA was inhibited by cyanamide, indicating that the acid is derived from an aldehyde dehydrogenase (ALDH)-catalyzed pathway. The overall rate of HNE metabolism was insensitive to inhibition of AR or ALDH, although inhibition of HNA formation by cyanamide led to a corresponding increase in the fraction of HNE metabolized by the GSH-linked pathway, indicating that ALDH-catalyzed oxidation competes with glutathione conjugation. These metabolic pathways may be the key regulators of the vascular effects of HNE and oxidized LDL.

Introduction

High concentrations of unsaturated aldehydes such as 4-hydroxy trans-2-nonenal (HNE), malonaldehyde (MDA) and acrolein [1], [2], [3] are generated during in vitro oxidation of low-density lipoprotein (LDL). These aldehydes form covalent adducts with apolipoprotein (apo) B [1], [2], [4], [5], [6], and trigger the uptake of LDL by scavenger receptors [6] located on vascular tissues, including vascular small muscle cells (VSMC) [1], [6]. Moreover, antibodies against protein–HNE and protein–MDA adducts stain atherosclerotic lesions [6], [7], [8], [9], and high titers of these antibodies are present in sera of human and animals with peripheral or coronary artery disease and in apoE-deficient mice [10], [11], [12]. The aldehydes generated during oxidation do not remain sequestered in the LDL particle, but diffuse to distal sites [13] generating epitopes that do not colocalize with apoB [6]. In addition, antibodies against protein aldehyde adducts also stain focal areas of neointima after balloon injury [14], and VSMC of arteries with giant cell arteritis [15]; and increased formation of lipoxidation products such as HNE and MDA has been reported for diabetic aorta [16]. Nevertheless, the contribution of these aldehydes to atherogenesis remains unclear.

The α,β-unsaturated aldehydes (alkenals and 4-hydroxyalkenals) are derived from the oxidation of ω-6-polyunsaturated fatty acids such as arachidonic, linolenic and linoleic acids [2], which are particularly abundant in LDL [17]. Due to the conjugated α,β-unsaturation, these aldehydes react avidly with cellular nucleophiles such as glutathione, cysteine, lysine and histidine side chains of proteins, and with DNA bases [2]. As a result, high concentrations of these aldehydes are cytotoxic to most cells, and non-cytotoxic concentrations cause profound changes in gene expression and cellular metabolism. The HNE, for instance, stimulates VSMC growth [18], and forms selective adducts with c-Jun N-terminal kinase, inducing its phosphorylation [19]. It also increases DNA binding activity of AP-1 [18], [20], production of reactive oxygen species (ROS) [20], and transforming growth factor-β [21], and inhibits the NF-κB/Rel system and the synthesis of tumor necrosis factor (TNF)-α [22]. These observations suggest that low steady-state generation of lipid-derived aldehydes mediates and sustains chronic inflammation. Indeed, it has been proposed that these aldehydes act as second messengers of ROS [2] to signal or indicate pro-oxidant states.

To understand how aldehydes derived from oxLDL regulate and alter VSMC function, it is essential to identify the processes involved in their metabolism and detoxification. However, little is known in regard to the mechanisms by which VSMC metabolize these aldehydes. The present study was, therefore, designed to identify the major biochemical pathways involved in the VSMC metabolism of HNE, which is one of the most abundant and toxic aldehydes generated during the oxidation of LDL [1], [2].

Section snippets

Materials

Cyanamide, 4-methyl pyrazole (4-MP), aldehyde dehydrogenase (ALDH), NAD, NADPH and glutathione (GSH) were purchased from Sigma Chemical Company. Sorbinil (CP-45643; (+)-(4S)-6-fluorospirol(chroman-4,4′-imidazolidine)-2,5’-dione) and tolrestat (Ay-27773; N-[6-methoxy-5-(trifloromethyl)-1-napthalenyl]thioxomethyl-glycine) were gifts from Pfizer and Ayerst, respectively. All other reagents were of the highest purity available.

Chemical synthesis

[4-³H]-HNE was synthesized as its dimethyl acetal with a specific activity of 75–100 mCi/mmol as described earlier [23]. Prior to the experiments, [³H]-HNE was released by the acid hydrolysis of the dimethyl acetal and purified by high-performance liquid chromatography (HPLC). Radiolabeled 1,4-dihydroxy-2-nonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), glutathionyl conjugate of HNE (GS-HNE) and its reduced form (GS-DHN) were synthesized using recombinant aldose reductase (AR) as described

HNE consumption

To examine HNE metabolism, T-75 flasks containing VSMC were used. The culture medium was removed and the cells were incubated with 5 or 10 μM [³H]-HNE in 10 ml KH buffer. Incubation of the cells with 5 or 10 μM HNE for 6 h did not cause a significant change in cell viability as determined by the MTT assay. For measuring the rate of HNE metabolism, aliquots were withdrawn at various times and the radioactivity in the medium was separated by HPLC. HNE remaining in the medium was determined by

Discussion

Despite extensive evidence implicating oxLDL and its products in regulating VSMC growth and function, the mechanisms by which these oxidants affect VSMC are not well understood. The observations that oxidation of LDL increases the formation of lipid-derived aldehydes [1], [2], [3] suggest that the generation and metabolism of these aldehydes may be an important determinant of the VSMC redox state. Surprisingly, little is known of the mechanism by which VSMC metabolize these aldehydes although

Acknowledgments

This work was supported in part by NIH grants HL55477, HL59378, DK36118, and AHA grant 0060350B. The authors also thank Todd Downes for his help in the preparation of the manuscript and figures.

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Identification of biochemical pathways for the metabolism of oxidized low-density lipoprotein derived aldehyde-4-hydroxy trans-2-nonenal in vascular smooth muscle cells

Abstract

Introduction

Section snippets

Materials

Chemical synthesis

HNE consumption

Discussion

Acknowledgments

Free. Radic. Biol. Med.

J. Biol. Chem.

J. Biol. Chem.

Free. Radic. Biol. Med.

Lancet

Biochem. Biophys. Res. Commun.

Atherosclerosis

J. Lipid Res.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Pharmacol.

Free. Radic. Biol. Med.

FEBS Lett.

Toxicol. Appl. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

FEBS Lett.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Free. Radic. Biol. Med.

J. Biol. Chem.