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NAD(P)H Oxidase-Dependent Regulation of CCL2 Production during Retinal Inflammation

¹From the VA Medical Center, Augusta, Georgia; and the ²Vascular Biology Center and the ³Departments of Ophthalmology, ⁴Pharmacology and Toxicology, and ⁵Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia.

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 LPS-treated 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 LPS-treated 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.

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				W. Zhang, B. Baban, M. Rojas, S. Tofigh, S. K. Virmani, C. Patel, M. A. Behzadian, M. J. Romero, R. W. Caldwell, and R. B. Caldwell Arginase Activity Mediates Retinal Inflammation in Endotoxin-Induced Uveitis Am. J. Pathol., August 1, 2009; 175(2): 891 - 902. [Abstract] [Full Text] [PDF]