Advanced glycation end product-induced activation of NF-kappaB is suppressed by alpha-lipoic acid in cultured endothelial cells.
Depletion of cellular antioxidant defense mechanisms and the generation of oxygen free radicals by advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic vascular complications. Here we demonstrate that incubation of cultured bovine aortic endothelial cells (BAECs) with AGE albumin (500 nmol/l) resulted in the impairment of reduced glutathione (GSH) and ascorbic acid levels. As a consequence, increased cellular oxidative stress led to the activation of the transcription factor NF-kappaB and thus promoted the upregulation of various NF-kappaB-controlled genes, including endothelial tissue factor. Supplementation of the cellular antioxidative defense with the natural occurring antioxidant alpha-lipoic acid before AGE albumin induction completely prevented the AGE albumin-dependent depletion of reduced glutathione and ascorbic acid. Electrophoretic mobility shift assays (EMSAs) revealed that AGE albumin-mediated NF-kappaB activation was also reduced in a time- and dose-dependent manner as long as alpha-lipoic acid was added at least 30 min before AGE albumin stimulation. Inhibition was not due to physical interactions with protein DNA binding, since alpha-lipoic acid, directly included into the binding reaction, did not prevent binding activity of recombinant NF-kappaB. Western blots further demonstrated that alpha-lipoic acid inhibited the release and translocation of NF-kappaB from the cytoplasm into the nucleus. As a consequence, alpha-lipoic acid reduced AGE albumin-induced NF-kappaB mediated transcription and expression of endothelial genes relevant in diabetes, such as tissue factor and endothelin-1. Thus, supplementation of cellular antioxidative defense mechanisms by extracellularly administered alpha-lipoic acid reduces AGE albumin-induced endothelial dysfunction in vitro.
Bierhaus
A
A
Department of Internal Medicine, University of Heidelberg, Germany.
Chevion
S
S
Chevion
M
M
Hofmann
M
M
Quehenberger
P
P
Illmer
T
T
Luther
T
T
Berentshtein
E
E
Tritschler
H
H
Müller
M
M
Wahl
P
P
Ziegler
R
R
Nawroth
P P
PP
D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000737:chemistry / Q000378:metabolism; D002451:Cell Compartmentation / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000494:pharmacology*; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism*; D010084:Oxidation-Reduction; D008063:Thioctic Acid / Q000494:pharmacology*; D013925:Thromboplastin / Q000235:genetics*
D000975:Antioxidants; D004268:DNA-Binding Proteins; D017127:Glycation End Products, Advanced; D016328:NF-kappa B; D008063:Thioctic Acid; D013925:Thromboplastin; D005978:Glutathione; D001205:Ascorbic Acid