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Megan Elise Capozzi, John S Penn; Production of inflammatory mediators by Müller cells and their effect on leukostasis are inhibited by Cytochrome P450-derived epoxygenated lipids. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5837.
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© ARVO (1962-2015); The Authors (2016-present)
Cytochrome P450 epoxygenases (CYPs) metabolize arachidonic acid and docosahexaenoic acid to epoxyeicosatrienoic acids (EETs) and epoxydocosapentaenoic acids (EDPs), respectively. EETs and EDPs exert multiple physiologic influences, including potent anti-inflammatory actions, but their tissue levels are limited by the soluble epoxide hydrolase enzyme (sEH). In diabetes and obesity, CYP and sEH levels are altered in various tissues, resulting in decreased EET and EDP levels. The purpose of this study was to determine the effect of modulating EET/EDP levels on palmitic acid (PA)-induced Müller cell production of inflammatory mediators (TNFα, IL-1β, and IL-6), and to determine whether increasing EET/EDP levels could inhibit inflammatory mediator-induced leukocyte adherence to human retinal microvascular endothelial cells (HRMEC).
Müller cells were treated with a general CYP inhibitor, SKF-525A, or PA (60µM and 250µM) for 24hrs, and expression of inflammatory mediators (TNFα, IL-1β, and IL-6) was measured by qRT-PCR. The effect of sEH knockdown by siRNA on inflammatory mediator expression was assessed after PA treatment. A parallel plate flow chamber technique was used to determine the effect of TNFα on leukocyte adherence to HRMEC; monolayers were treated with TNFα (1ng/ml) in the presence or absence of 11,12-EET (0.5µM) or 19,20-EDP (0.5µM) with the sEH inhibitor, AUDA (10µM).
SKF-525A treatment increased Müller cell expression of TNFα, IL-1β, and IL-6 by 4.1-fold (p=0.05), 2.9-fold (p<0.001) and 1.4-fold (p<0.001), respectively. Similarly, 250µM PA induced their expression by 3.6-fold (p<0.001), 64% (p=0.003) and 2.4-fold (p<0.001), respectively. sEH knockdown significantly inhibited both constitutive and induced expression of these mediators. TNFα treatment of HRMEC increased leukocyte adherence by 3.3-fold (p<0.001), and 11,12-EET + AUDA and 19,20-EDP + AUDA inhibited TNFα-induced leukocyte adherence by 48% and 50%, respectively.
Retinal PA and TNFα are increased in diabetics and promote inflammation in retinal Müller and endothelial cells. The pro-inflammatory effects of these diabetes-relevant insults appear to be at least partly CYP-dependent, and can be inhibited by increasing EETs and EDPs in culture. Future work will determine the EET/EDP-dependency of the retinal inflammatory responses in vivo.
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