In:
Journal of Vascular Research, S. Karger AG, Vol. 49, No. 2 ( 2012), p. 144-159
Abstract:
We investigate the cell signal transduction pathway protein kinase C (PKC) and the role of NADPH subunits in the process of TNF-α-induced endothelial apoptosis. Human umbilical vein endothelial cells (HUVEC) were treated with one of these: 1 m 〈 i 〉 M 〈 /i 〉 PKC β 〈 sub 〉 2 〈 /sub 〉 inhibitor CGP53353, 10 m 〈 i 〉 M 〈 /i 〉 PKC δ inhibitor rottlerin, combination CGP53353 with rottlerin, 3 ×10 〈 sup 〉 –4 〈 /sup 〉 〈 i 〉 M 〈 /i 〉 NADPH oxidase inhibitor apocynin, 5 × 10 〈 sup 〉 –6 〈 /sup 〉 〈 i 〉 M 〈 /i 〉 NADPH oxidase peptide inhibitor gp91ds-tat. The apoptosis process was assessed by Hoechst 33342 stain, flow cytometry and Western blot analysis, while intracellular reactive oxygen species (ROS) production was detected by 2,7’-dichlorodihydrofluorescein diacetate (DCFH-DA). The NADPH oxidase subunit gene and protein expression were assessed by quantitative real-time PCR and Western blot analysis, respectively. TNF-α significantly induced HUVEC apoptosis and ROS production, accompanying with dramatic upregulation of NADPH oxidase subunits: NOX2/gp91 〈 sup 〉 phox 〈 /sup 〉 , NOX4, p47 〈 sup 〉 phox 〈 /sup 〉 and p67 〈 sup 〉 phox 〈 /sup 〉 , whereas these enhancements were abolished by the treatment with PKC inhibitors. High TNF-α level exposure induces HUVEC apoptosis, as well as a ROS generation increase via the PKC β 〈 sub 〉 2 〈 /sub 〉 -dependent activation of NADPH oxidase. Although the PKC δ pathway may enhance TNF-α-induced HUVEC apoptosis, it does not involve the ROS pathway. Upregulation of expression of NADPH subunits is important in this process, which leads to a new target in antioxidative therapy for vascular disease prevention.
Type of Medium:
Online Resource
ISSN:
1018-1172
,
1423-0135
Language:
English
Publisher:
S. Karger AG
Publication Date:
2012
detail.hit.zdb_id:
1482726-8
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