Plasmalogens In The Regulation Of Retinal Vascular Development: Design Of A Mouse Model With Chemical Inhibition Of iPLA2

Abstract

Purpose: : Plasmalogens represent a particular class of phospholipids characterized by a vinyl ether bond in sn-1 position of glycerol and by its abundance in nervous tissues and in the heart. Plasmalogen deficiency in mice leads to developmental abnormalities in retinal vasculature (Acar et al, ARVO 2007 E-Abstract 2978) and to persistent hyaloid arteries (DAPAT^-/- mice, Rodemer et al, Hum Mol Genet, 2003). We hypothesize that plasmalogens regulate retinal vascular development through the liberation of polyunsaturated fatty acids (PUFA) by a plasmalogen-selective calcium-independent phospholipase A2 (iPLA2) (Acar et al, ARVO 2009 E-Abstract 2925). The aim of this work was to establish a mouse model in which iPLA2 is inhibited by chemical treatment.

Methods: : In order to inhibit iPLA2, we have tested on C57BL/6 mice the effects of three different inhibitors known for their powerful effect on iPLA2 enzymes: Bromoenolactone (BEL), Heparan Sulfate and FKGK11. After adapting a cPLA2 Assay Kit to measure iPLA2 activity, the in vitro and in vivo activities of iPLA2 were determined in total protein extracts of brain or heart tissues.

Results: : We have determined that a minimum of 200 µg of total proteins is necessary to obtain reliable and interpretable results when using the cPLA2 assay kit. Two inhibitors have been finally adopted to validate the inhibition of iPLA2 enzymes in the model: BEL and Heparan Sulfate. The more interesting in vitro results were obtained with BEL. Indeed, when using BEL, the percentage of inhibition of iPLA2 was about of 50% at 25 µM and reached its maximum (85%) at 135 µM. We have also obtained an additional weak inhibition of cPLA2 from 100 µM. Optimal doses for in vivo tests were set between 25 and 100 µM. In these conditions, we have obtained 30% of inhibition of iPLA2, after an hour of treatment in C57BL/6 mice. Since we have previously demonstrated that 30% of retinal plasmalogens belongs to ethanolamine family (PlsEtn) (Acar and al, Exp Eye Res 2007), our studies were further focused on the activity of retinal PlsEtn specific iPLA2 (PlsEtn-PLA2).

Conclusions: : This mouse model of chemical inhibition of iPLA2 and retinal PlsEtn-PLA2 may be useful in understanding the cellular and molecular mechanisms by which plasmalogens may regulate retinal vascular development during early life.

Keywords: lipids • metabolism • retinal neovascularization