Increased synthesis of leukotrienes in the mouse model of diabetic retinopathy.

Early inflammatory changes in diabetic retinopathy include leukocyte adherence to the microvasculature (i.e., leukostasis), alterations in vascular permeability, and enhanced expression of proinflammatory molecules such as NF-κB, COX-2, iNOS, and ICAM1.1–6 It is hypothesized that this inflammatory state leads to damage to the retinal microvasculature, including degeneration of the capillaries with loss of the surrounding pericytes, hallmark findings in nonproliferative diabetic retinopathy.7–9 Recently, we implicated the leukotrienes, 5-lipoxygenase metabolites of arachidonic acid, as mediators of inflammation in diabetic retinopathy.10 In comparison to diabetic control mice, retinas from diabetic mice deficient in 5-lipoxygenase developed less capillary degeneration and loss of pericytes and less leukostasis, superoxide production, and activation of NF-κB.10 Synthesis of leukotrienes requires the sequential action of several enzymes11 (Fig. 1). Arachidonic acid is released by the calcium-dependent activation of cytosolic phospholipase A2α (cPLA2α). Arachidonic acid is then metabolized to 5-hydroperoxyeicosatetraenoic acid (5-HpETE) and, subsequently, to LTA4 by 5-lipoxygenase and its associated 5-lipoxygenase activating protein. The detection of LTA4 is complicated by its extremely short half-life (<3 seconds).12 Production of LTA4 is the first committed step in leukotriene biosynthesis. LTA4 is rapidly metabolized to LTB4 by LTA4 hydrolase or, alternatively, to LTC4 by LTC4 synthase, which requires conjugation with glutathione. LTC4 can be further metabolized to LTD4 and LTE4; collectively, these metabolites constitute the cysteinyl leukotrienes. Nonenzymatic hydrolysis of LTA4 can also occur with the formation of isomers of Δ6-trans-LTB4 and 5,6-diHETE. LTB4 is a known leukocyte attractant and binds to BLT1 and BLT2 receptors. Signaling through BLT1 has been linked to ROS generation, cytokine activation, and apoptosis.13–15 Activation of the cysteinyl leukotriene receptors CysLT1, CysLT2, and CysLTE mediates changes in vascular permeability and modulates inflammation.16–20 Leukotriene synthesis is increased in many inflammatory disease states, including asthma, colitis, arthritis, and CNS injury.16,21–26 Figure 1. Synthesis of leukotrienes. Although cells of the myeloid lineage are well-recognized sources of leukotriene production, an alternative synthetic pathway for leukotrienes involves transcellular metabolism.27,28 In this two-cell process of leukotriene synthesis, the donor cell (believed to be a bone marrow-derived cell, such as the neutrophil or the macrophage) generates LTA4, which then passes transcellularly to the recipient cell, where it is further metabolized to downstream leukotrienes, depending on the enzymatic repertoire of the recipient cell. In a model of neuronal ischemia in the brain, glial cells demonstrated the enzymatic capability to produce leukotrienes by transcellular biosynthesis.29 It is hypothesized that the local recipient cell generation of leukotrienes may cause focal areas of prolonged inflammation and tissue injury.27,28,30 We investigated the effects of chronic exposure to elevated glucose concentrations on the generation of leukotrienes by mouse bone marrow cells, retinal glial cells, and retinal microvascular endothelial cells as potential mediators of chronic inflammation and endothelial cell injury in the diabetic retina.