4-hydroxy-2-nonenal and sorbinil

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 [(3)H]-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.

Topics: Aldehyde Reductase; Aldehydes; Alkenes; Animals; Aorta; Cells, Cultured; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Glutathione; Imidazoles; Imidazolidines; Lipoproteins, LDL; Male; Mass Spectrometry; Muscle, Smooth, Vascular; Naphthalenes; Rats; Rats, Sprague-Dawley

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is an important feature of atherosclerosis, restenosis, and hypertension. Although multiple mediators of VSMC growth have been identified, few effective pharmacological tools have been developed to limit such growth. Recent evidence indicating an important role for oxidative stress in cell growth led us to investigate the potential role of aldose reductase (AR) in the proliferation of VSMCs. Because AR catalyzes the reduction of mitogenic aldehydes derived from lipid peroxidation, we hypothesized that it might be a potential regulator of redox changes that accompany VSMC growth. Herein we report several lines of evidence suggesting that AR facilitates/mediates VSMC growth. Stimulation of human aortic SMCs in culture with mitogenic concentrations of serum, thrombin, basic fibroblast growth factor, and the lipid peroxidation product 4-hydroxy-trans-2-nonenal (HNE) led to a 2- to 4-fold increase in the steady-state levels of AR mRNA, a 4- to 7-fold increase in AR protein, and a 2- to 3-fold increase in its catalytic activity. Inhibition of the enzyme by sorbinil or tolrestat diminished mitogen-induced DNA synthesis and cell proliferation. In parallel experiments, the extent of reduction of the glutathione conjugate of HNE to glutathionyl-1,4-dihydroxynonene in HNE-exposed VSMCs was decreased by serum starvation or sorbinil. Immunohistochemical staining of cross sections from balloon-injured rat carotid arteries showed increased expression of AR protein associated with the neointima. The media of injured or uninjured arteries demonstrated no significant staining. Compared with untreated animals, rats fed sorbinil (40 mg. kg(-1). d(-1)) displayed a 51% and a 58% reduction in the ratio of neointima to the media at 10 and 21 days, respectively, after balloon injury. Taken together, these findings suggest that AR is upregulated during growth and that this upregulation facilitates growth by enhancing the metabolism of secondary products of reactive oxygen species.

Topics: Aldehyde Reductase; Aldehydes; Angioplasty, Balloon; Animals; Aorta; Carotid Artery Injuries; Carotid Stenosis; Cell Division; Cells, Cultured; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Fibroblast Growth Factor 2; Gene Expression Regulation, Enzymologic; Glutathione; Hemostatics; Humans; Imidazoles; Imidazolidines; Lipid Peroxidation; Male; Muscle, Smooth, Vascular; Naphthalenes; Rats; Rats, Sprague-Dawley; Recurrence; RNA, Messenger; Thrombin; Tritium

Giant cell arteritis (GCA) is a systemic vasculitis preferentially affecting large and medium-sized arteries. Inflammatory infiltrates in the arterial wall induce luminal occlusion with subsequent ischemia and degradation of the elastic membranes, allowing aneurysm formation. To identify pathways relevant to the disease process, differential display-PCR was used. The enzyme aldose reductase (AR), which is implicated in the regulation of tissue osmolarity, was found to be upregulated in the arteritic lesions. Upregulated AR expression was limited to areas of tissue destruction in inflamed arteries, where it was detected in T cells, macrophages, and smooth muscle cells. The production of AR was highly correlated with the presence of 4-hydroxynonenal (HNE), a toxic aldehyde and downstream product of lipid peroxidation. In vitro exposure of mononuclear cells to HNE was sufficient to induce AR production. The in vivo relationship of AR and HNE was explored by treating human GCA temporal artery-severe combined immunodeficiency (SCID) mouse chimeras with the AR inhibitors Sorbinil and Zopolrestat. Inhibition of AR increased HNE adducts twofold and the number of apoptotic cells in the arterial wall threefold. These data demonstrate that AR has a tissue-protective function by preventing damage from lipid peroxidation. We propose that AR is an oxidative defense mechanism able to neutralize the toxic effects of lipid peroxidation and has a role in limiting the arterial wall injury mediated by reactive oxygen species.

Topics: Aldehyde Reductase; Aldehydes; Animals; Apoptosis; Benzothiazoles; Chimera; Enzyme Inhibitors; Free Radical Scavengers; Giant Cell Arteritis; Humans; Imidazoles; Imidazolidines; Lipid Peroxidation; Mice; Mice, SCID; Phthalazines; RNA, Messenger; Temporal Arteries; Thiazoles; Up-Regulation; Vasculitis

Hydrogen peroxide (H2O2) or 4-hydroxy-2,3-trans-nonenal (HNE) treatment of rat vascular smooth muscle cells (A7r5) caused induction of aldose reductase mRNA. Induction was dose (10-100 microM H2O2, 1-10 microM HNE) and time dependent, reaching a maximum (three- to fourfold) after 7-12 h. Treatment of cells with actinomycin D confirmed de novo synthesis of aldose reductase mRNA. H2O2-induced expression was prevented by catalase but unaffected by Desferal, indicating that metal catalyzed degradation of peroxide was not involved. Induction of enzymatically active aldose reductase by H2O2 and HNE was confirmed using Western blotting and enzyme assays. Aldose reductase can metabolize several aldehyde compounds including HNE, a major toxic product of lipid peroxidation. Inclusion of Sorbinil, an aldose reductase inhibitor, in toxicity assays resulted in a significant (twofold) enhancement of HNE-mediated killing of A7r5 cells, suggesting a protective role of aldose reductase against HNE-induced cell death. These data indicate that the induction of aldose reductase during oxidative stress might represent an important cellular antioxidant defense mechanism.

Topics: Aldehyde Reductase; Aldehydes; Animals; Arsenites; Catalase; Cells, Cultured; Deferoxamine; Enzyme Induction; Hydrogen Peroxide; Imidazoles; Imidazolidines; Muscle, Smooth; Oxidative Stress; Rats; RNA; RNA, Messenger; Sodium Compounds

Kinetic and structural changes in recombinant human aldose reductase (AR) due to modification by S-nitrosoglutathione (GSNO) were investigated. Incubation of the enzyme with 10-50 microM GSNO led to a time- and concentration-dependent inactivation of the enzyme, with a second-order rate constant of 0.087 +/- 0.009 M-1 min-1. However, upon exhaustive modification, 30-40% of the enzyme activity was retained. The non-inactivated enzyme displayed a 2-3-fold change in Km for NADPH and Km fordl-glyceraldehyde, whereas the Km for the lipid peroxidation product, 4-hydroxy-2-trans nonenal (HNE), was comparable to that of the untreated enzyme. The residual activity of the enzyme after GSNO treatment was less sensitive to inhibition by the active site inhibitor sorbinil or to activation by sulfate. Significantly higher catalytic activity was retained when the enzyme was modified in the presence of NADPH, suggesting relatively low reactivity of the E-NADPH complex with GSNO. The modification site was identified using site-directed mutants in which each of the solvent-exposed cysteines of the enzyme was replaced individually by serine. The mutant C298S was insensitive to GSNO, whereas the sensitivity of the mutants C303S and C80S was comparable to that of the wild-type enzyme. Electrospray ionization mass spectroscopy of the GSNO-modified enzyme revealed a major modified species (70% of the protein) with a molecular mass that was 306 Da higher than that of the untreated enzyme, which is consistent with the addition of a single glutathione molecule to the enzyme. The remaining 30% of the protein displayed a molecular mass that was not significantly different from that of the native enzyme. No nitrosated forms of the enzyme were observed. These results suggest that inactivation of AR by GSNO is due to the selective formation of a single mixed disulfide between glutathione and Cys-298 located at the NADP(H)-binding site of the enzyme.

Topics: Aldehyde Reductase; Aldehydes; Disulfides; Enzyme Activation; Enzyme Inhibitors; Glutathione; Glutathione Disulfide; Glyceraldehyde; Humans; Imidazoles; Imidazolidines; Iodoacetates; Iodoacetic Acid; Kinetics; Mass Spectrometry; Mutagenesis, Site-Directed; NADP; Nitroso Compounds; Placenta; Recombinant Proteins; S-Nitrosoglutathione; Sulfates

Free radicals have extremely short half-lives and they readily oxidize lipids and initiate an autocatalytic chain reaction of lipid peroxidation, which leads to the formation of lipid peroxides. The lipid peroxides undergo degradation to form metastable lipid aldehydes such as 4-hydroxynonenal (HNE). We have shown earlier that under hyperglycemia, lipid peroxides increase; and aldose reductase, an enzyme that reduces glucose to sorbitol, efficiently reduces HNE. The purpose of the present studies was thus to investigate the role of HNE in hyperglycemic cataract and understand the mechanism(s) of its prevention by antioxidants and aldose reductase inhibitors. HNE and hyperglycemic cataract were developed by culturing rat lenses in TC-199 medium containing 50 microM HNE and 50 mM glucose, respectively. The effect of an anti-oxidant, trolox, and an aldose reductase inhibitor, sorbinil, on the progression of HNE and hyperglycemic cataract, evaluated by digital image analysis, was followed for 8 and 9 days, respectively. In lenses cultured with HNE, the decrease in transmitted light was 43, 65, and 87% on Days 3, 5, and 8, respectively. Trolox ameliorated the HNE cataract, whereas sorbinil accelerated the progression of HNE cataract and prevented the progression of hyperglycemic cataract. It is concluded that HNE formed under hyperglycemia may play a pivotal role in diabetic cataractogenesis.

Topics: Aldehyde Reductase; Aldehydes; Animals; Cataract; Chromans; Cross-Linking Reagents; Enzyme Inhibitors; Image Enhancement; Imidazoles; Imidazolidines; Rats