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W. Zhang, M. Bartoli, M. Rojas, N.-T. Tsai, B. Lilly, R. W. Caldwell, R. B. Caldwell; Regulation of CCL2 Production via NAD(P)H Oxidase-Modulated Stat3 Activation in Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5891.
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© ARVO (1962-2015); The Authors (2016-present)
Both vascular inflammation and oxidative stress are critically involved in the pathogenesis of diabetic retinopathy, retinopathy of prematurity (ROP), and uveitis. CCL2 [monocyte chemoattractant protein (MCP)-1], plays an important role in vascular inflammation by inducing leukocyte recruitment. This study was undertaken to determine if and how NAD(P)H oxidase, a major source of reactive oxygen species in vascular cells, regulates CCL2 production in vascular inflammation.
These experiments were performed in the mouse model for oxygen-induced retinopathy (OIR) and in cultured endothelial cells (ECs).
Expression of TNF-α, VEGF and CCL2 increased during relative hypoxia in the OIR mouse retina. Since it is known that TNF-α induces CCL2 expression in EC via activation of NF-kB and p38MAPK, we determined if NAD(P)H oxidase regulates TNF-α-induced CCL2 expression via NF-kB and p38MAPK. Studies using RT-PCR and ELISA demonstrated that inhibition of NAD(P)H oxidase completely blocked TNF-α-induced CCL2 expression in human ECs. However, inhibition of NAD(P)H oxidase did not block TNF-α-induced p38MAPK activation and only partially blocked TNF-α-induced NF-kB activation, suggesting that other mediators serve as downstream targets for NAD(P)H oxidase. Analysis of the promoter region of CCL2 disclosed a conserved STAT/GAS binding site. Detecting STATs phosphorylation revealed that TNF-α robustly induced STAT3 phosphorylation and weakly activated STAT1 but had no effect on STAT5 or STAT6. Stattic, a specific inhibitor for STAT3, dose dependently blocked TNF-α-induced STAT3 phosphorylation and also abolished TNF-α-induced CCL2 production. Moreover, inhibition of NAD(P)H oxidase completely blocked TNF-α-induced STAT3 activation. Parallel studies using VEGF revealed that the same signaling pathway is responsible for VEGF-induced CCL2 production.
This research demonstrates that TNF-α and VEGF stimulate CCL2 production through NAD(P)H oxidase regulation of the STAT3 pathway. Modulation of NAD(P)H oxidase/STAT3 pathway may provide therapeutic benefit for the treatment of retina vascular inflammation.
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