Increased Expression and Activity of 12-Lipoxygenase in Oxygen-Induced Ischemic Retinopathy and Proliferative Diabetic Retinopathy Implications in Retinal Neovascularization Mohamed Al-Shabrawey, 1,2,3,4 Rene Mussell, 1 Khalid Kahook, 1 Amany Tawfik, 1 Mohamed Eladl, 4 Vijay Sarthy, 5 Julian Nussbaum, 2 Ahmed El-Marakby, 1 Sun Young Park, 6 Zafer Gurel, 6 Nader Sheibani, 6 and Krishna Rao Maddipati 7 OBJECTIVE—Arachidonic 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 endothe- lial cell proliferation and migration. The goal of this study was to investigate whether 12-LOX plays a role in retinal neovasculari- zation (NV). RESEARCH DESIGN AND METHODS—Experiments were performed using retinas from a murine model of oxygen-induced ischemic retinopathy (OIR) that was treated with and without the LOX pathway inhibitor, baicalein, or lacking 12-LOX. We also analyzed vitreous samples from patients with and without proliferative diabetic retinopathy (PDR). Western blotting and RT-PCR were used to assess the expression of 12-LOX, vascular endothelial growth factor (VEGF), and pigment epithelium– derived factor (PEDF). Liquid chromatography–mass spectrome- try was used to assess the amounts of HETEs in the murine retina and human vitreous samples. The effects of 12-HETE on VEGF and PEDF expression were evaluated in Müller cells (rMCs), primary mouse retinal pigment epithelial cells, and astrocytes. RESULTS—Retinal NV during OIR was associated with in- creased 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 epi- thelium. Only rMCs and astrocytes showed increased VEGF ex- pression by 12-HETE. CONCLUSIONS—12-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. Diabetes 60:614–624, 2011 R etinal neovascularization (NV) is a vision- threatening complication of ischemic retinopa- thy that develops in various retinal disorders, including diabetic retinopathy and retinopathy of prematurity (ROP). Retinal NV is controlled by a bal- anced production of pro- and antiangiogenic factors, in- cluding vascular endothelial growth factor (VEGF) and pigment epithelium–derived factor (PEDF), respectively (1). However, under some pathological conditions, in- cluding diabetic retinopathy and ROP, this balance is dis- rupted 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 A 2 (5), arach- idonic acid is metabolized via different pathways, in- cluding 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 diox- ygenases 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 arach- idonic 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 con- tribute to tumor angiogenesis via a VEGF-dependent path- way (20) and to stimulate endothelial cell mitogenesis (7,8) and tube formation (21). From the 1 Department of Oral Biology and Anatomy, Medical College of Geor- gia, Augusta, Georgia; the 2 Ophthalmology and Vision Discovery Institute, Medical College of Georgia, Augusta, Georgia; the 3 Department of Ophthal- mology, College of Medicine, King Saud University, Riyadh, Saudi Arabia; the 4 Department of Anatomy, Mansoura College of Medicine, Mansoura, Egypt; 5 Northwestern University, Chicago, Illinois; the 6 Department of Oph- thalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin; and the 7 Department of Pathology, Wayne State University, Detroit, Michigan. Corresponding author: Mohamed Al-Shabrawey, malshabrawey@mcg.edu. Received 4 January 2010 and accepted 21 November 2010. DOI: 10.2337/db10-0008 Ó 2011 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. 614 DIABETES, VOL. 60, FEBRUARY 2011 diabetes.diabetesjournals.org ORIGINAL ARTICLE Downloaded from http://diabetesjournals.org/diabetes/article-pdf/60/2/614/398410/614.pdf by guest on 04 January 2023