Advanced Glycation End Product-Induced Activation of NF-κB is Suppressed by α-Lipoic Acid in Cultured Endothelial Cells

Abstract

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.

Advanced glycation end-products: a review

“…The NFkB is a free radical sensitive transcription factor that modulates gene transcription for endothelin-1, VCAM-1, tissue factor and thrombomodulin [12]. It has also been linked to states of anti-oxidant depletion induced by AGE, with a reduction in glutathione, vitamin C and nitric oxide [66]. Further evidence of an interaction between AGE and the NF-kB pathway can be shown from in vitro studies using hyaluronic acid, which is a glycosaminoglycan polymer that inhibits AGE-induced activation of NF-kB and NF-kB regulated release of IL-1a, IL-6 and TNFa [67].…”

Regulation of Inducible Nitric Oxide Synthase Expression in Advanced Glycation End Product–Stimulated RAW 264.7 Cells

“…Preparation of BSA and AGEs. AGEs were prepared according to previously described methods (29). Briefly, BSA and AGEs were prepared by incubation of 3.14 mg/ml BSA in the presence or absence of 200 mmol/l D-glucose and 0.5 mmol/l sodium azide in PBS (pH 7.4) at 37°C for 12 weeks with constant agitation.…”

Rage gene promoter polymorphisms and diabetic retinopathy in a clinic-based population from South India

“…RAGE is expressed in many of the cell types, such as the endothelial cells, monocytes, and lymphocytes, including the beta cells of the pancreas. RAGE-mediated signaling leads to the activation of transcription factors, such as NF-kB, AP-1, and STAT-1, 5,6 the adhesion molecules VCAM, ICAM, and tissue factor, 7,8 which promote a procoagulant state in the microcapillaries of the retina. This results in a hypoxic state that leads to the initiation of the angiogenic process in proliferative DR.…”