Several mechanisms have been suggested to

Several mechanisms have been suggested to account for the causal association of ICAM-1 induction and leukocyte adhesion with DR, including oxidative stress [16], NF-κB [17], PKC [18], and bioactive lipids [19]. Our previous studies highlighted the role of bioactive lipid metabolites derived from 12/15-lipoxygenase (LO) in pathogenesis of the inflammatory response in early DR. We have shown an increase in the retinal 12/15-LO expression and activity, evident by elevated levels of 12/15-LO-derived hydroxyeicosatetraenoic Riociguat (HETE) locally; in the vitreous of patients with DR [20], diabetic mouse retinas [21], and human retinal endothelial cells (HRECs) incubated with high glucose [22]. Additionally, we have reported a reduction in diabetes-induced retinal ICAM-1 expression by the pharmacological inhibition of 12/15-LO [21]. Furthermore, our very recent studies have demonstrated the ability of intravitreally injected 12-HETE to compromise endothelial barrier function in the retina associated with the induction of a pro-inflammatory phenotype (increased in retinal ICAM-1 expression and leukocyte adhesion) [22,23]. As a further support for the role of 12/15-LO in early DR, we have shown by using fluorescein angiography (FA) and retinal albumin leakage assay a significant reduction in retinal barrier dysfunction in diabetic mice lacking global expression of 12/15-LO compared to diabetic wild type (WT) mice [22]. Two distinct mammalian 12/15-Lipoxygenases have been characterized on the basis of their products from arachidonic acid (AA); 15-lipoxygenase (15-LO) in humans [24] and rabbits [25], and its orthologue the “leukocyte-type” known as 12-lipoxygenase (12-LO) in pig, rat, and mouse [26]. Leukocyte 12-LO and 15-LO are highly related in their enzymological characteristics as well as primary structures, and both are collectively called 12/15-LO [27]. A variety of cells, including vascular and myeloid lineage cells such as endothelial cells, smooth muscle cells, platelets, and monocytes/macrophages, express 12/15-LO [28]. By looking at the 12/15-LO activity in different cell types, it has been shown that 12/15-LO may have opposing effects. For instance, overexpression of 12/15-LO in endothelial cells enhances atherogenic responses [29], while overexpression of 12/15-LO in monocytes/macrophages protects against atherogenesis [30]. These observations raised the possibility that different cellular expression of 12/15-LO may have different effects on pathogenesis of DR and this has not been previously addressed. Therefore, the current study aimed to characterize the relative contribution of retinal endothelial versus monocytic/macrophagic 12/15-LO to inflammatory responses in DR. To achieve this goal, we first evaluated the changes in circulating 12/15-LO activity by measuring its derived metabolites in the plasma of diabetic WT mice compared to non-diabetic controls. This was followed by comparing the in vitro endothelium-leukocytes interaction between leukocytes isolated from 12/15-LO−/− versus those isolated from WT mice. Finally, we examined the effects of knocking down or inhibiting endothelial 12/15-LO on diabetes-induced HREC activation and ICAM-1 expression.
Materials and methods
Results
Discussion The novel and central finding of the current study is that endothelial 12/15-LO rather than the monocytic/macrophagic 12/15-LO has a critical role in hyperglycemia-induced leukocyte adhesion and retinal endothelial barrier dysfunction. The following evidence, in addition to what we have published for the elevated levels of 12/15-LO metabolites locally under diabetic conditions, support this conclusion: (a) No significant change in the systemic plasma levels of primary metabolites derived from the activity of 12/15-LO, including 12- and 15-HETE, in STZ-diabetic mice. (b) Leukocytes from 12/15-LO−/− mice displayed a similar increase in adhesion to activated endothelial cells as did leukocytes from WT mice. (c) Abundant proteins of 12-LO and 15-LO were detected in HRECs, while it was undetected (15-LO) or hardly detectable (12-LO) in human monocyte-like U937 cells. (d) Inhibition of endothelial 12/15-LO in HRECs attenuated hyperglycemia-induced leukocyte adhesion and ICAM-1 expression, a well-known identified important molecule for leukocyte adhesion in DR.