Abstract
Purpose:
Cytochrome P450 epoxygenases (CYPs) convert arachidonic acid to epoxyeicosatrienoic acids (EETs). EETs act by both autocrine and paracrine mechanisms and have demonstrated potent anti-inflammatory activity. EET action is limited by hydration of its epoxide group by soluble epoxide hydrolase (sEH) and conversion to a less active diol. In diabetic animals, CYP expression and EET production have been shown to be reduced in various tissues, however the role of CYPs in the diabetic retina has never been studied. The purpose of this study was to determine the effect of diabetes-relevant stimuli on CYP expression in human Müller cells (HMC) and human retinal astrocytes (HRA) and to determine the effect of EETs on the inflammatory response of human retinal microvascular endothelial cells (HRMEC).
Methods:
HMC and HRA were cultured in normal glucose (5.5mM), high glucose (25.5mM), or a mannitol osmotic control (25.5mM). CYP2C8 and CYP2C9 expression were measured by qRT-PCR. HRMEC were treated with TNFα in the presence or absence of 11,12-EET (0.5µM) and a sEH inhibitor (AUDA; 1.0µM). NFkB translocation was assessed by western blot. ICAM-1, VCAM-1 and MCP-1 expression were measured by qRT-PCR.
Results:
Twenty-four hours after glucose treatment, CYP2C8 expression was inhibited by 33.7% (p=0.0058) and CYP2C9 by 53.2% (p=0.0037), in HMC. High glucose had no effect on CYP expression in HRA. In HRMEC, translocation of the NFkB p65 subunit to the nucleus was stimulated by TNFα and inhibited by 11,12-EET and AUDA treatment. 11,12-EET and AUDA inhibited TNFα-induced ICAM-1 expression by 30.7% (p=0.0097), VCAM-1 expression by 62.1% (p=0.0011) and MCP-1 expression by 30.9% (p=0.0073) in HRMEC.
Conclusions:
High glucose reduced the expression of EET-producing CYP2C enzymes in Müller cells, but not retinal astrocytes. TNFα-induced inflammation in HRMEC was inhibited by the 11,12-EET CYP product. Future studies will determine the effect of EET treatment in rodent models of non-proliferative diabetic retinopathy.
Keywords: 499 diabetic retinopathy •
506 eicosanoids •
557 inflammation