Effect of homocysteinylation of low density lipoproteins on lipid peroxidation of human endothelial cells

Abstract

Homocysteine-thiolactone (HcyT) is a toxic product whose synthesis is directly proportional to plasma homocysteine (Hcy) levels. Previous studies demonstrated that the interaction between HcyT and low density lipoproteins (LDL) induces the formation of homocystamide-LDL adducts (Hcy-LDL). Structural and functional alterations of Hcy-LDL have been described and it has been suggested that homocysteinylation could increase atherogenicity of LDL. Oxidative damage of endothelial cells (EC) is considered to be a critical aspect of the atherosclerotic process. To further investigate the molecular mechanisms involved in the atherogenicity of homocysteinylated LDL, we studied the effect of interaction between Hcy-LDL and EC on cell oxidative damage, using human aortic endothelial cells (HAEC) as experimental model. Homocysteinylation of LDL was carried out by incubation of LDL, isolated from plasma of healthy normolipemic subjects, with HcyT (10-100 microM). In our experimental conditions, homocysteinylation treatment was not accompanied by oxidative damage of LDL. No modifications of apoprotein structure and physico-chemical properties were observed in Hcy-LDL with respect to control LDL (c-LDL), as evaluated using the intrinsic fluorescence of tryptophan and the probe Laurdan incorporated in lipoproteins. Our results demonstrated that Hcy-LDL incubated at 37 degrees C for 3 h with HAEC, induced an oxidative damage on human EC with a significant increase of lipid hydroperoxides in cells incubated with Hcy-LDL with respect to cell incubated with c-LDL. The compositional changes were associated with a significant decrease viability in cells treated with Hcy-LDL. The relationship between the levels of -SH groups of LDL and the oxidative damage of HAEC has been demonstrated. These results suggest that Hcy-LDL exert a cytotoxic effect that is likely related to an increase in lipid peroxidation and oxidative damage of EC.

Factors Affecting S-Homocysteinylation of LDL Apoprotein B

“…At least 9 free sulfhydryl groups (ϪSH) in apoB-100 primary structure have been reported that could potentially bind plasma free aminothiols (13 ). Because lysyl residues of apoB could react in vivo with plasma HcyT by an amide bond, the number of these apoprotein sulfhydryl groups could increase considerably, thus increasing the number of sites that may be bound by plasma aminothiols (9 ). Even if we did not measure protein N-homocysteinylation concentrations, a 50 g/L LDL solution can be calculated to contain ϳ1 mol/L of Hcy-N-apoprotein, as recently reported by Jakubowski (29 ).…”

“…Even at low concentrations (up to 10 mol/ L), Hcy is cytotoxic and induces cell injury and oxidative damage in cultured cells, with generation of reactive oxidative species, formation of superoxide and hydrogen peroxide, and increased lipid peroxidation product concentrations (10,11 ). LDL incubation with 100 mol/L HcyT for 2 h was reported to cause the N-homocysteinylation of ϳ10% of apoB100 lysyl residues (9 ). Assuming a relative molecular mass (M r ) of ϳ500 000 for apoB and ϳ200 lysyl residues per apoB molecule (12 ), ϳ20 new ϪSH groups are introduced in the sequence of apoprotein, thus tripling the number of "free sulfydryl" groups of native protein, reported to be 9 (13 ).…”

May modifications of human plasma proteins stimulated by homocysteine and its thiolactone induce changes of hemostatic function of plasma in vitro?

“…For example, homocysteinylated molecules gain biotoxic features like LDL molecules, which after interaction with HTL can induce endothelial lipid peroxidation (Ferretti et al 2004) or peroxynitrite production by human aortic endothelial cells (Ferretti et al 2006). Coagulation factor Va is also S-homocysteinylated (Undas et al 2001) and this modification could play a role in the modulation of hemostasis process.…”

Homocysteine and metabolic risk factors in individuals with family history of premature ischemic stroke

“…In an experimental model, it has been demonstrated that homocysteinylated LDL intensifies the cytotoxic effect associated with increased lipid peroxidation and oxidative vascular endothelial cell damage. 22,23 The vascular endothelium damaged by hyperhomocysteinemia initiates inflammation and development of atherosclerotic lesions. 24 In women, we observed an association between lower HDL-C and elevated Hcy levels, which might increase the probability of HDL modification by Hcy.…”