NAD(P)H oxidase regulates CCL2 production during retinal inflammation

¹ Vascular Biology Center, Medical College of Georgia, Augusta, Georgia, United States
² Ophthalmology, Medical College of Georgia, Augusta, Georgia, United States
³ Pharmacology and Toxicology, Medcial College of Georgia, Augusta, Georgia, United States
⁴ Vascular Biology Center, Medical College of Georgia, 1120 15 St, Augusta, Georgia, 30912, United States

^* To whom correspondence should be addressed. E-mail: rcaldwel{at}mail.mcg.edu.

	Abstract

Purpose: CCL2 plays an important role in vascular inflammation by inducing leukocyte recruitment and activation. We have found that blockade of NAD(P)H oxidase blocks leukocyte adhesion to retinal vessels during diabetes and uveitis. Here we assess the role of NAD(P)H oxidase in CCL2 production. 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-kappa B activity was detected by reporter gene assay. Kinase phosphorylation was determined by immunoblotting. Results: Expression of CCL2 was increased in retinas from all three mouse models. The effect was strongest in the LPS-treated mice, with a peak mRNA increase at 3h. This increase was abrogated by administration of NAD(P)H oxidase inhibitor apocynin. Apocynin also blocked CCL2 production in endothelial cells (ECs), retinal microglia, and Muller cells stimulated with TNF-alpha, VEGF, or LPS. Studies using human ECs demonstrated that TNF-alpha-induced CCL2 production was also inhibited by the NAD(P)H oxidase inhibitor DPI, the antioxidant NAC or the superoxide scavenger Tiron, further indicating that the inhibition is through the NAD(P)H/ROS pathway. Analysis of downstream signals showed that inhibition of NAD(P)H oxidase partially inhibited NF-kappa B activation, but did not reduce CCL2 mRNA stability or prevent TNF-alpha-induced phosphorylation of p38MAPK. However, TNF-alpha-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-kappa B are involved in this signaling pathway.

Key Words: gene expression, signal transduction, retinal inflammation, CCL2, NAD(P)H oxidase

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