TY - JOUR
T1 - Generation of reactive oxygen intermediates, activation of NF-κB, and induction of apoptosis in human endothelial cells by glucose
T2 - Role of nitric oxide synthase?
AU - Du, Xueliang
AU - Stockklauser-Färber, Karin
AU - Rösen, Peter
N1 - Funding Information:
This work was supported by the Ministerium für Frauen, Familie und Gesundheit der Bundesrepublik Deutschland and the Wissenschaftsministerium des Landes NRW, the Deutsche Forschungsgemeinschaft, Bonn, and the “Klinische Zellbioloogie und Biophysik” e.V., Düsseldorf.
PY - 1999/10
Y1 - 1999/10
N2 - 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. Copyright (C) 1999 Elsevier Science Inc.
AB - 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. Copyright (C) 1999 Elsevier Science Inc.
KW - Apoptosis
KW - Diabetes mellitus
KW - Endothelium
KW - Free radicals
KW - Hyperglycemia
KW - Nitric oxide synthase
KW - Peroxynitrite
KW - Reactive oxygen intermediates
KW - Transcription factor NF-κB
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U2 - 10.1016/S0891-5849(99)00079-9
DO - 10.1016/S0891-5849(99)00079-9
M3 - Article
C2 - 10515579
AN - SCOPUS:0344850195
SN - 0891-5849
VL - 27
SP - 752
EP - 763
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
IS - 7-8
ER -