Generation of reactive oxygen intermediates, activation of NF-κB, and induction of apoptosis in human endothelial cells by glucose: role of nitric oxide synthase?

Abstract

Exposure to high glucose causes characteristic dysfunction and morphologic changes of the endothelium. To study the underlying mechanisms of glucotoxicity, human endothelial cells (HUVECs) were isolated from umbilical veins and cultivated under hyperglycemic conditions (10–30 mM) for up to 72 h. The generation of reactive oxygen intermediates (ROIs) was determined by histochemical staining of the cells by dichlorodihydrofluorescein. Activation of the transcription factor nuclear factor-kappa B (NF-κB) family was analyzed by the electromobility shift assay and by histochemical staining of the cells with rhodamine-labelled consensus sequences of activated NF-κB. Apoptotic cells were identified by morphologic analysis and DNA fragmentation. Incubation of HUVECs with high glucose led to rapid increase in the generation of ROIs. After an incubation of 2 to 6 h, NF-κB became activated, with the maximum at 4 h. Exposure of HUVECs to high glucose for up to 72 h caused a significant induction of apoptosis in HUVECs. The increased generation of ROIs, activation of apoptosis, and induction of apoptosis were also observed in cells incubated with 3-O-methyl-D-glucose, a glucose derivative that is taken up by the cells but not metabolized. Generation of ROIs, activation of NF-κB, and induction of apoptosis were not only prevented by antioxidants (thioctic acid, tocopherol, superoxide dysmutase-mimetic), but also by l-nitroarginine. These observations indicate that high glucose leads to an increase in generation of ROIs, an activation of NF-κB, and an induction of apoptosis by a glucose-specific and NO synthase-dependent mechanism. Our data suggest that peroxynitrite, which is rapidly formed from nitric oxide and superoxide anions, is the mediator of the cytotoxic effects of high glucose on endothelial cells. Because the induction of apoptosis by glucose was prevented by an antisense nucleotide to the p65NF-κB binding site, we assume that the ROI-mediated activation of NF-κB plays an important role for induction of apoptosis by glucose.

Introduction

Exposure of endothelium to hyperglycemia can cause characteristic dysfunction of endothelium as observed in diabetes [1]. In addition to changes in the proliferation of endothelial cells and an increased synthesis of matrix proteins and growth factors [2], [3], [4], [5], [6], an impairment of endothelium-dependent vasodilatation has been described [1], [7], [8], [9]. The induction of apoptosis by high glucose can be taken as another indicator of changes in the regulation of gene transcription by high glucose [10], [11], [12], [13]. The mechanisms underlying the endothelial dysfunction and the thrombogenic transformation of endothelium in diabetes are not fully understood. Identifying the mechanisms by which high glucose may lead to endothelial dysfunction may help to develop strategies to prevent it from occurring and to develop adjunctive therapeutic interventions to delay the development of vascular complications in diabetes.

Transcription factors of the nuclear factor-kappa B (NF-κB/Rel) family form dimeric complexes that control the expression of a plethora of genes involved in inflammation and proliferation [14], [15], [16]. It has already been shown that activation of NF-κB plays an important role for the activation of genes encoding tumor necrosis factor-α, interleukin-1β, macrophage colony stimulating factor, the monocyte chemoattractant protein 1, tissue factor, vascular adhesion molecule 1, and intercellular adhesion molecule 1 [14], [16]. Because oxidized low-density lipoproteins, advanced glycation end products (AGE), and high glucose have been shown to activate NF-κB in blood and endothelial cells [17], [18], [19], [20], [21], [22], it is reasonable to suggest that this mechanism may also be of large importance for the structural and functional alterations in the vessel walls in diabetes and may contribute to the high thrombogenicity of the diabetic vessel wall.

Why high glucose is able to activate NF-κB is not very well understood. We have hypothesized recently that an increased oxidative stress is a major cause because there is much evidence that NF-κB is activated by ROIs and, on the other hand, that the generation of ROIs is increased by diabetes [23], [24], [25]. It has also been shown that the activation of NF-κB by AGE is closely associated with the receptor-mediated stimulation of ROI formation [19]. Here we present evidence that high glucose directly activates NF-κB and that this process is inhibited by antioxidants, suggesting that high glucose induces a pathophysiologic cascade of reactions that begins with the formation of ROIs and the activation of NF-κB and may finally explain the thrombogenic transformation observed in diabetic patients. However, the activation of NF-κB was not only inhibited by antioxidants and superoxide dismutase mimetic, but also by inhibition of endothelial nitric oxide synthase (NOS). Our observations suggest that it is the reaction product of nitric oxide (NO) and superoxide anions—peroxynitrite—that contributes to the activation of NF-κB and the thrombogenic transformation of endothelium in hyperglycemia.

Further, it is not known whether the activation of NF-κB is related to the induction of apoptosis observed in diabetic vessels and endothelial cells cultivated in high glucose [10], [11], [12], [13]. The role of NF-κB is discussed controversially: pro- and antiapoptotic effects of NF-κB activation have been shown [26], [27], [28]. We here demonstrate that the induction of apoptosis by high glucose is prevented under similar conditions as the activation of NF-κB and by specific p65 NF-κB antisense oligodeoxynucleotides.