NAD(P)H oxidase-dependent regulation of CCL2 production during retinal inflammation

Wenbo Zhang, Modesto Rojas, Brenda Lilly, Nai Tse Tsai, Tahira Lemtalsi, Gregory I. Liou, Robert W. Caldwell, Ruth B. Caldwell

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

PURPOSE. CCL2 plays an important role in vascular inflammation by inducing leukocyte recruitment and activation. The authors had previously found that the blockade of NAD(P)H oxidase in turn blocks leukocyte adhesion to retinal vessels during diabetes and uveitis. In this study, the role of NAD(P)H oxidase in CCL2 production was assessed. METHODS. Studies were performed in three mouse models with lipopolysaccharide (LPS)-induced uveitis, ischemic retinopathy, and streptozotocin diabetes and in cytokine-and LPStreated cells. CCL2 mRNA and protein expression were measured by quantitative PCR and ELISA. NF-_B activity was detected by reporter gene assay. Kinase phosphorylation was determined by immunoblotting. RESULTS. Expression of CCL2 was increased in the retinas of all three mouse models. The effect was strongest in the LPStreated mice, with a peak mRNA increase at 3 hours. This increase was abrogated by administration of the NAD(P)H oxidase inhibitor apocynin. Apocynin also blocked CCL2 production in endothelial cells (ECs), retinal microglia, and Müller cells stimulated with TNF-_, VEGF, or LPS. Studies using human ECs demonstrated that TNF-_-induced CCL2 production was also inhibited by the NAD(P)H oxidase inhibitor DPI, the antioxidant N-acetyl-L-cysteine, or the superoxide scavenger Tiron, further indicating that inhibition occurs through the NAD(P)H/ROS pathway. Analysis of downstream signals showed that inhibition of NAD(P)H oxidase partially inhibited NF-_B activation but did not reduce CCL2 mRNA stability or prevent TNF-_-induced phosphorylation of p38MAPK. However, TNF-_-induced Akt phosphorylation was blocked, and inhibiting Akt dramatically decreased CCL2 production. CONCLUSIONS. NAD(P)H oxidase activity is required for CCL2 production during retinal vascular inflammation. Akt and NF-_B are involved in this signaling pathway.

Original languageEnglish (US)
Pages (from-to)3033-3040
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume50
Issue number6
DOIs
StatePublished - 2009

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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    Zhang, W., Rojas, M., Lilly, B., Tsai, N. T., Lemtalsi, T., Liou, G. I., Caldwell, R. W., & Caldwell, R. B. (2009). NAD(P)H oxidase-dependent regulation of CCL2 production during retinal inflammation. Investigative Ophthalmology and Visual Science, 50(6), 3033-3040. https://doi.org/10.1167/iovs.08-2676