Increased expression and activity of 12-lipoxygenase in oxygen-induced ischemic retinopathy and proliferative diabetic retinopathy: implications in retinal neovascularization

OBJECTIVEArachidonic acid is metabolized by 12-lipoxygenase (12-LOX) to 12-hydroxyeicosatetraenoic acid (12-HETE) and has an important role in the regulation of angiogenesis and endothelial cell proliferation and migration. The goal of this study was to investigate whether 12-LOX plays a role in retinal neovascularization (NV).RESULTSRetinal NV during OIR was associated with increased 12-LOX expression and 12-, 15-, and 5-HETE production. The amounts of HETEs also were significantly higher in the vitreous of diabetic patients with PDR. Retinal NV was markedly abrogated in mice treated with baicalein or mice lacking 12-LOX. This was associated with decreased VEGF expression and restoration of PEDF levels. PEDF expression was reduced in 12-HETE–treated rMCs, astrocytes, and the retinal pigment epithelium. Only rMCs and astrocytes showed increased VEGF expression by 12-HETE.CONCLUSIONS12-LOX and its product HETE are important regulators of retinal NV through modulation of VEGF and PEDF expression and could provide a new therapeutic target to prevent and treat ischemic retinopathy.Retinal neovascularization (NV) is a vision-threatening complication of ischemic retinopathy that develops in various retinal disorders, including diabetic retinopathy and retinopathy of prematurity (ROP). Retinal NV is controlled by a balanced production of pro- and antiangiogenic factors, including vascular endothelial growth factor (VEGF) and pigment epithelium–derived factor (PEDF), respectively (1). However, under some pathological conditions, including diabetic retinopathy and ROP, this balance is disrupted by enhanced production of proangiogenic and/or downregulation of antiangiogenic factors (2,3).Arachidonic acid metabolites, which are known as eicosanoids, are involved in regulating angiogenesis (4). Once released by cytosolic phospholipase A₂ (5), arachidonic acid is metabolized via different pathways, including the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 pathways. Angiogenesis has been shown to be promoted by the metabolic products of COX2, prostaglandins (6) and the products of the lipoxygenase system, leukotrienes, and hydroxyeicosatetraenoic acids (HETEs) (7,8). LOXs are a group of closely related dioxygenases that catalyze the stereospecific oxygenation of arachidonic acid and other polyunsaturated fatty acids (PUFAs) and are classified as 5-, 8-, 12-, or 15-LOX, according to the site of oxygen insertion within arachidonic acid.Three types of 12-LOX have been characterized: platelet, leukocyte, and epidermal 12-LOX (9), which are detected in various cell types, including smooth muscle cells (SMCs) (10), endothelial cells (10,11), and glial cells (12). The major product of 12-LOX metabolism of arachidonic acid, 12-HETE has a role in various biological processes, including atherogenesis, cancer cell growth, and neuronal apoptosis (13,14). In addition, 12-HETE has proinflammatory effects (15,16) and has been implicated in diabetic vascular complications (13). For example, high glucose treatment increases 12-HETE production in vascular endothelial cells and SMCs, and this increase is linked to vascular endothelial growth factor (VEGF) upregulation (17,18) and leukostasis (19) in the intracellular adhesion molecule-1–dependent pathway (15). Similarly, 12-HETE has been shown to contribute to tumor angiogenesis via a VEGF-dependent pathway (20) and to stimulate endothelial cell mitogenesis (7,8) and tube formation (21).VEGF and PEDF are identified as key angiogenic factors whose altered production contributes to the development of retinal NV. They induce opposite effects in the retina, which causes vasculopathies associated with diabetic retinopathy and ROP. Although VEGF has angiogenic and permeability effects that were shown to be mediated via oxidative stress (22,23) and inflammatory pathways (24), PEDF elicits antiangiogenic and antipermeability effects in part through antioxidant (25,26) and anti-inflammatory mechanisms (27). There are multiple cellular sources for the growth factors involved in retinal NV. Müller cells (rMCs) are known to express several modulators of angiogenesis by responding to hypoxia or hyperglycemia and releasing VEGF (28,29). They also are shown to have an antiangiogenic background attributed to PEDF secretion (30). Moreover, VEGF and PEDF expression in rMCs are altered by a high glucose concentration, which contributes to retinal NV in diabetic retinopathy (31).The role of lipoxygenases in general, and 12-LOX in particular, in the development of retinal NV has not been well investigated. The goal of this study was to explore the changes in 12-LOX expression and activity during retinal NV and to determine whether targeting 12-LOX activity impacts retinal NV perhaps through changes in the level of angiogenic factors.The current study presents, for the first time, that oxygen-induced ischemic retinopathy (OIR) and proliferative diabetic retinopathy (PDR) are associated with increased 12-LOX expression and activity. Inhibition of the LOX pathway or 12-LOX deletion significantly abrogated retinal NV and VEGF expression, while preserving retinal PEDF levels during OIR.