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

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.

Mitogenic Responses of Vascular Smooth Muscle Cells to Lipid Peroxidation-derived Aldehyde 4-Hydroxy-trans-2-nonenal (HNE)

“…Serum-starved VSMC were stimulated for 24 h with the indicated concentrations of GS-DHN-ester in the presence of 20 g/ml of RLIP76 Ab1 and the % proliferation of VSMC was determined by MTT assay as described under "Experimental Procedures." diovascular tissues (37,38,41). Glutathione conjugates generated in vivo are extruded from peripheral tissues.…”

“…At the indicated time points the culture media was applied to HPLC. Peak 1 eluted at fractions 22-25 corresponded to GS-HNE/GS-DHN, peak 2 eluted at fractions 28 -31 corresponded to GS-HNE-ester/GS-DHN-ester, and peak 3 eluted at fractions [35][36][37][38] that incubation with HNE increased cell growth. A similar increase in growth was observed when the cells were incubated with GS-HNE-and GS-DHN-esters.…”

“…In VSMC, HNE is conjugated to glutathione to form GS-HNE and a portion of this conjugate is reduced to GS-DHN by AR (37,38). To distinguish whether GS-HNE or GS-DHN is mitogenic, we examined how inhibition of AR would affect the mitogenicity of the conjugates.…”

“…Regulate VSMC Growth-Conjugation of HNE with glutathione is followed by reduction and both reduced and oxidized conjugates are actively extruded from cells (37,38). Hence, one way to enhance the cellular effects of the conjugates is to increase their intracellular residence time by inhibiting their extrusion.…”

“…Our previous studies show that VSMC transform HNE into multiple metabolites (37,38). These include direct oxidation and reduction of HNE to 4-hydroxynonanoic acid and 1,4-dihydroxynonene (DHN), respectively.…”