Endothelial but not Leukocytic 12/15-Lipoxygenase Contributes to Retinal Leukostasis in Diabetic Retinopathy

Abstract

Purpose : Leukocyte-endothelial cell adhesion is an essential step for the initiation of retinal inflammatory response prior to clinical signs of DR. Our previous studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory response in DR. In the current study we aimed to disscet out the relative contribution of retinal endothelial 12/15-LO versus monocytic/macrophagic 12/15-LO in DR.

Methods : We first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of 6-months Streptozotocin-induced diabetic male mice (n=6-8) using liquid chromatography coupled with mass-spectrometry (LC-MS) to evaluate changes in circulating 12/15-LO activity. This is followed by comparing the in vitro mouse endothelium-leukocytes interaction between leukocytes isolated from 12/15-LO knockout (KO) versus those isolated from wild type (WT) mice using myeloperoxidase (MPO) assay. Finally, we examined the effects of knocking down or inhibiting endothelial 12/15-LO on diabetes-induced endothelial cell activation and ICAM-1 expression, an important molecule for leukocyte adhesion in DR (n=4). Two-tailed Student’s t-test was used for statistical analysis.

Results : Analysis of plasma bioactive lipids’ heat map revealed that the activity of circulating 12/15-LO did not altered during diabetes as evident by non-significant changes in plasma levels of major metabolites derived from 12/15-lipoxygenation of different PUFAs, including linoleic acid (13-HODE), arachidonic acid (12- and 15- HETEs), eicosapentaenoic acid (12- and 15- HEPEs), or docosahexaenoic acid (17-HDoHE). Moreover, leukocytes from 12/15-LO KO mice displayed a similar increase in adhesion to high glucose (HG)-activated endothelial cells as do leukocytes from WT mice. Furthermore, abundant proteins of 12-LO and 15-LO were detected in human retinal endothelial cells (HRECs), while it was undetected (15-LO) or hardly detectable (12-LO) in human monocyte-like cells. Inhibition or knock down of endothelial 12/15-LO in HRECs significantly (P<0.05) reduced HG-induced expression of ICAM-1.

Conclusions : Our data support our hypothesis that that endothelial 12/15-LO rather than myeloid lineage 12/15-LO is implicated in hyperglycemia-induced activation of retinal endothelial cells. This may facilitate the development of more precisely targeted treatment strategies.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.