Homocysteinylation of low-density lipoproteins (LDL) from subjects with Type 1 diabetes: effect on oxidative damage of human endothelial cells.
BACKGROUND
Homocysteine (Hcy) is an independent risk factor for cardiovascular disease (CVD). Individuals with Type 1 and Type 2 diabetes are more susceptible to the effects of homocysteine than non-diabetic subjects. The interaction between homocysteine-thiolactone (Hcy-thiolactone), a reactive product of Hcy, and low-density lipoproteins (LDL) induces the formation of homocystamide-LDL adducts (Hcy-LDL) and it has been suggested that homocysteinylation could increase atherogenicity of lipoproteins.


OBJECTIVE
The aim of the study was to compare the effect of in vitro homocysteinylation of LDL isolated from healthy control subjects (C-LDL) and from Type 1 diabetic patients (DM-LDL) and to investigate the effect of homocysteinylated LDL (Hcy-C-LDL and Hcy-DM-LDL) on peroxynitrite production of endothelial cells.


METHODS
The in vitro homocysteinylation of LDL isolated from control (n = 12) and DM subjects (n = 12) was carried out by incubating lipoproteins with Hcy-thiolactone. The reaction was verified by quantifying the increase in sulphydryl groups (-SH groups) in Hcy-LDL with respect to control LDL. Control and homocysteinylated LDL were incubated with human aortic endothelial cells (HAEC) in culture. Peroxynitrite production in cells treated in different experimental conditions was assayed by a fluorimetric method.


RESULTS
The increase in -SH groups after incubation with homocysteine was greater in LDL from diabetic subjects compared with LDL from control subjects (P < 0.001). In addition, peroxynitrite production from HAEC incubated with Hcy-LDL from diabetic patients was greater than after incubation with Hcy-LDL from control subjects and untreated LDL from diabetic patients (P < 0.001).


CONCLUSIONS
These results show that LDL from diabetic patients is more susceptible to in vitro homocysteinylation than LDL from non-diabetic individuals and demonstrate that the compositional changes in Hcy-LDL from diabetic subjects have cytotoxic effects on human endothelial cells.
Ferretti
G
G
Istituto of Biochimica, Facoltà di Medicina e Chirurgia, Università Politecnica delle Marche, Ancona, Italy. g.ferretti@univpm.it
Bacchetti
T
T
Rabini
R A
RA
Vignini
A
A
Nanetti
L
L
Moroni
C
C
Mazzanti
L
L
D000328:Adult; D001011:Aorta / Q000378:metabolism; D050197:Atherosclerosis / Q000150:complications / Q000378:metabolism*; D003922:Diabetes Mellitus, Type 1 / Q000150:complications / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism*; D006710:Homocysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008077:Lipoproteins, LDL / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D030421:Peroxynitrous Acid / Q000096:biosynthesis*; D011837:Radiation-Protective Agents / Q000494:pharmacology
D008077:Lipoproteins, LDL; D011837:Radiation-Protective Agents; D006710:Homocysteine; D030421:Peroxynitrous Acid; C007957:homocysteine thiolactone