oa 12/15-lipoxygenase: A Novel Therapeutic Target In Diabetic Retinopathy

Background: Breakdown of blood-retinal barrier (BRB) and subsequent hyperpermeability is a cardinal feature of early diabetic retinopathy (DR) and leading cause of blindness. Fatty acid metabolism is implicated in several biological functions via multiple signaling pathways including the synthesis of bioactive metabolites. Our previous studies established 12/15-lipoxygenase (12/15-LOX)-derived 12- and 15- hydroxyeicosatetraenoic acids or HETEs as proangiogenic mediators during DR via disrupting the retinal levels of vascular endothelial growth factor and pigment epithelium derived factor (PEDF). Purpose: 1) To screen the impact of high glucose (HG) treatment on the levels of bioactive lipids including 12/15-LOX metabolites in cultured human retinal endothelial cells (HREC), 2) To test the impact of 12/15-LOX deletion on BRB function and explore the underlying mechanism in particular the role of NADPH oxidase as a major source of oxidative stress during DR. Material and Methods: Liquid chromatography-mass spectrometry (LC/MS) was used in a comprehensive lipidomic screen of HRECs treated with or without HG (30mM D-glucose). Effect of 12- or 15-HETE (0.1 M) on leukostasis and angiogenesis (tube formation) was examined in HRECs transfected with siRNA against the catalytic subunit of NADPH oxidase (NOX2) or the scrambled siRNA or treated with or without the NADPH oxidase inhibitor (apocynin, 30°M). Human leukocytes (PMNs) labeled with lipophilic fluorescent probe were used for leukostasis assay. Migration assay was performed using the Electrical Cell Impedance Sensor (ECIS). Retinal vascular changes were examined by fluorescein angiogram (FA) and Optical Coherence tomogram (OCT) in streptozotocin-induced diabetic wild type (WT) or 12/15-LOX-knockout mice and in mice received intraocular injection with 12-HETE or 12-HETE with PEDF. Western blotting, immunofluorescence and multiplex system were used to test the changes in the levels of inflammatory markers. Results: Out of 126 bioactive lipids screened, 70 were undetected, 47 un-significantly changed, and 9 metabolites were significantly up-regulated by HG compared to osmotic control (5mM D-glucose+25 mM L-glucose). Intriguingly, six metabolites among the 9-increased are the products of 12/15-LOX pathway (15-HETE, 11-HETE, 8, 15-DiHETE, 12-HETE, 17-HDoHE, and 13-HODE). Of these 15-HETE has the highest fold increase (p value= 0.004). Thereafter, we pursued to determine whether the altered metabolites of 12/15 LOX play a role in BRB dysfunction during diabetes. Retinal vascular leakage was significantly reduced in diabetic mice deficient in 12/15-LOX as evidence by FA and less albumin leakage compared with diabetic wild-type littermates. Furthermore, intravitreal injection of 12-HETE per se in normal WT mice resulted in a marked increases in the vascular leakage and expression of inflammatory markers such as ICAM-1, VCAM-1, CD45 as well as the NOX2 compared to vehicle-injected control or to mice received both 12-HETE and PEDF. In vitro, 12/15-HETEs induced HREC leukostasis, migration, and tube formation. These effects were inhibited by the concomitant use of apocynin and by silencing NOX2. Conclusion: Retinal endothelial 12/15-LOX is activated by hyperglycemia causing overproduction of 12- and 15-HETEs. These metabolites are implicated in the pro-angiogenic, -oxidative and -inflammatory effects of hyperglycemia in HREC. Thus, targeting this signaling system represents an attractive therapeutic strategy to prevent and treat DR.