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Ahmed S Ibrahim, Khaled Hussein, Amany M Tawfik, Nasser Rizk, Sylvia B Smith, Mohamed Al-Sayed Al-Shabrawey; 12/15-Lipoxygenase Gene Deletion Attenuates Blood-Retinal Barrier Dysfunction During Diabetic Retinopathy: Contribution of ER Stress. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5852.
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© ARVO (1962-2015); The Authors (2016-present)
Diabetic retinopathy (DR) is characterized by the development of intraretinal microvascular abnormalities. However there are still notable gaps in the understanding of underlying biological processes. Bioactive Lipid metabolites derived from 12/15-lipoxygenase -(LOX) are attracting great interest as disease-related molecules with little attention in the context of early DR. Therefore, the aims of the current study are: 1)To screen the impact of hyperglycemia on the levels of bioactive lipids, including 12/15-LOX metabolites, in human retinal endothelial cells (HREC). 2)To test the impact of 12/15-LOX deletion on blood retinal barrier (BRB) function and explore the underlying mechanism in particular the role of Endoplasmic reticulum (ER) stress which is known to play a pathogenic role in DR.
Liquid chromatography-mass spectrometry (LC/MS) screen of bioactive lipids in HRECs treated with or without high glucose has been used. In vitro Barrier function assay was performed using the Electrical Cell Impedance Sensor (ECIS). Retinal vascular changes were examined by fluorescein angiogram (FA) in living mice and by Western blot analysis for total retinal albumin.
Out of 126 bioactive lipids screened, 70 were undetected, 47 whose levels un-significantly changed, and 9 metabolites were significantly up-regulated under hyperglycemic condition. 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 (2.4±0.4). Next, we investigated the direct effects of 15-HETE on HREC monolayer. The barrier function of HRECs has been significantly reduced by 15-HETE treatment and was associated with increased ER stress compared to vehicle treated cells. Thereafter, we determined whether 12/15-LOX plays a causative role in the pathogenesis of BRB dysfunction during diabetes using 12/15-LOX(-/-) mice. Diabetic 12/15-LOX(-/-) mice had less vascular hyper-fluorescence on FA and less albumin leakage compared with diabetic wild-type littermates. This improvement in barrier function seen in 12/15-LOX(-/-) mice was associated with decreased ER stress sensor protein, CHOP.
12/15-LOX/ER stress interplay is implicated in breakdown of BRB and represents a potential therapeutic target to treat microvascular dysfunction in DR.
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