Targeting of 12/15-Lipoxygenase in retinal endothelial cells, but not in monocytes/macrophages, attenuates high glucose-induced retinal leukostasis

Abstract

Aims Our previous studies have established a role for 12/15-lipoxygenase (LO) in mediating the inflammatory response in diabetic retinopathy (DR). However, the extent at which the local or systemic induction of 12/15-LO activity involved is unclear. Thus, the current study aimed to characterize the relative contribution of retinal endothelial versus monocytic/macrophagic 12/15-LO to inflammatory responses in DR. Materials & Methods We first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of streptozotocin (STZ)-induced diabetic mice using liquid chromatography coupled with mass-spectrometry (LC-MS) to evaluate changes in circulating 12/15-LO activity. This was 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 the 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. Results Analysis of plasma bioactive lipids’ heat map revealed that the activity of circulating 12/15-LO was not altered by diabetes as evident by no significant changes in the 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 U937 cells. Inhibition or knock down of endothelial 12/15-LO in HRECs blocked HG-induced expression of ICAM-1, a well-known identified important molecule for leukocyte adhesion in DR. Conclusion Our data support that endothelial, rather than monocytic/macrophagic, 12/15-LO has a critical role in hyperglycemia-induced ICAM-1 expression, leukocyte adhesion, and subsequent local retinal barrier dysfunction. This may facilitate the development of more precisely targeted treatment strategies for DR.

The Role of Inflammation in Diabetic Retinopathy

“…It is important to consider that increased leukocyte trapping/ leukostasis parallels the increase in blood glucose/dyslipidemia (146,147) but this has been attributed by some authors to changes in the endothelium rather than the circulating leukocytes (148). As detailed above, hyperglycemia activates circulating leukocytes which are then liable to adhere to vessels walls (149,150).…”

Bone Morphogenetic Protein (BMP)4 But Not BMP2 Disrupts the Barrier Integrity of Retinal Pigment Epithelia and Induces Their Migration: A Potential Role in Neovascular Age-Related Macular Degeneration

“…Effects of BMP2 or 4 on barrier function of ARPE-19 were evaluated by monitoring changes in Trans-Cellular Electrical Resistance (TER). Normalized TER was recorded by Electric Cell-substrate Impedance Sensing (ECIS ® Zθ (theta)) instrument (Applied Biophysics Inc, Troy, NY, USA) as previously described [27][28][29]. Briefly, a 96-wells arrays (catalog # 96W20idf PET, Applied Biophysics Inc) were coated with 100 µM cysteine (50 µL) for 30 min then with 0.02% gelatin (50 µL) for another 30 min.…”

Role of the 12-lipoxygenase pathway in diabetes pathogenesis and complications

“…Intravitreal injection of 12(S)-HETE compromises the endothelial barrier in the retina leading to inflammatory changes (Ibrahim et al, 2015b). Recent studies indicate that retinal endothelial cells, but not monocytes or macrophages, are key cells linked to 12/15-LOX-related retinal effects (Ibrahim et al, 2017). Not all of the effects leading to DR in the capillary bed are due to 12/15-LOX, since other studies also suggest a role of 5-LOX (Gubitosi-Klug et al, 2017) in the mouse.…”