Anti–Inflammatory Effect of PPAR in Human Retinal Vascular Endothelial Cells

Abstract

Abstract: : Purpose: Early diabetic retinopathy has been recognized as a low–grade chronic inflammatory disease. We have previously demonstrated that n6 fatty acids (linoleic_18:2n6 and arachidonic_20:4n6 ) have a pro–inflammatory effect and the major n3 polyunsaturated fatty acid (PUFA) in the retina, docosahexaenoic acid (DHA_22:6n3), has a pronounced anti–inflammatory effect on hRVE cells. Fatty acids can bind and activate transcription factors, such as the peroxisome proliferator–activated receptor (PPAR) family (PPARα, ß and γ). All three isoforms of PPARs have been shown to modulate immune response. The purpose of this study was to address the role of PPAR activation in anti–inflammatory effect of n3 polyunsaturated fatty acids in hRVE cells. Methods: PPARα, ß and γ mRNA levels were determined by Reverse transcription followed by Real time PCR. The expression levels of PPARα, ß and γ, intercellular adhesion molecules–1 (ICAM–1) and vascular adhesion molecules (VCAM)–1 were assayed by Western Blotting. Lipofectamine transfection with previously described plasmid, pMN–rPPARα–LBD, that express PPARα–LBD fused to the Gal4 DNA binding domain and with p–MHTK100x4–Luc reporter that contains four binding sites for the Gal4–DNA–binding domain was used for PPARα transactivation assay. Results: hRVE cells express all three PPAR isoforms with PPARß being the most abundant. PPARα agonist WY14643 dose dependently inhibited VEGF165 and TNFα induced VCAM–1 and ICAM–1 expression in hRVE cells. PPARγ agonists troglitazone and Azelaoyl PAF, and PPARß agonist GW51016 had no effect. PPARα transactivation assay was used to determine to determine the effect of fatty acids on PPARα activity in hRVE cells. PPARα agonist WY14643 induced 9 fold increase in luciferase activity. Exogenous free fatty acids such as palmitic_16:0, linoleic_18:2n6 and arachidonic_{20:4n6 ,} and DHA_22:6n3 all increased luciferase activity to a similar degree. Conclusions: Activation of PPARα, but not PPARß and γ specifically prevent VEGF and TNFα induced adhesion molecules expression, suggesting that PPARα agonist could have a beneficial effect in preventing inflammation associated with early stage diabetic retinopathy. However, the differential effect of n3 and n6 fatty acids on inflammatory adhesion molecules expression in hRVE cells can not be explained by PPARα activation alone.

Keywords: inflammation • vascular cells • diabetic retinopathy