March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Increased Retinal Endothelial Cell Permeability By 12/15-lipoxygenase-derived Eicosanoids: A Possible Role Of NADPH Oxidase
Author Affiliations & Notes
  • Amira O. Othman
    Oral Biology and Anatomy,
    Georgia Health Sciences University, Augusta, Georgia
  • Saif Ahmad
    Ophthalmology,
    Georgia Health Sciences University, Augusta, Georgia
  • Sylvia Megyerdi
    Oral Biology and Anatomy,
    Georgia Health Sciences University, Augusta, Georgia
  • Karishma Choksi
    Oral Biology and Anatomy,
    Georgia Health Sciences University, Augusta, Georgia
  • Ali Behzadian
    Vascular Biology,
    Georgia Health Sciences University, Augusta, Georgia
  • Gregory I. Liou
    Ophthalmology,
    Georgia Health Sciences University, Augusta, Georgia
  • Krishna Maddipati
    Pathology, Wayne State University, Detroit, Michigan
  • Mohamed Al-Shabrawey
    Oral Biology and Anatomy,
    Ophthalmology,
    Georgia Health Sciences University, Augusta, Georgia
  • Footnotes
    Commercial Relationships  Amira O. Othman, None; Saif Ahmad, None; Sylvia Megyerdi, None; Karishma Choksi, None; Ali Behzadian, None; Gregory I. Liou, None; Krishna Maddipati, None; Mohamed Al-Shabrawey, None
  • Footnotes
    Support  AHA00104 and VDI-GHSU
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 5401. doi:
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      Amira O. Othman, Saif Ahmad, Sylvia Megyerdi, Karishma Choksi, Ali Behzadian, Gregory I. Liou, Krishna Maddipati, Mohamed Al-Shabrawey; Increased Retinal Endothelial Cell Permeability By 12/15-lipoxygenase-derived Eicosanoids: A Possible Role Of NADPH Oxidase. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5401.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Our previous studies demonstrated that NADPH oxidase-derived reactive oxygen species (ROS) and 12/15-lipoxygenase (12/15-LOX)-derived eicosanoids (12- and 15-hydroxyeicosatetraenoic acids or HETEs) contribute to retinal neovascularization. The purpose of this study was to evaluate the hypothesis that 12/15-LOX-derived HETEs alter retinal endothelial cell (REC) barrier via NADPH oxidase dependent mechanism.

Methods: : The effect of 12- and 15-HETE (0.1 and 0.5 μM) on bovine REC barrier was evaluated by measuring FITC-dextran flux across the monolayers grown on porous membranes and by electrical cell-substrate impedance sensing (ECIS) to measure changes in transendothelial electrical resistance (TER). ROS production in BRECs treated with or without 12-HETE was measured by dihydroethedium (DHE) and dichlorofluorescein fluorescence reaction in the presence or absence NADPH oxidase specific inhibitors (apocynin, 30 μM).Western blotting (WB) was also used to evaluate the expression of NADPH oxidase catalytic subunit NOX2. The levels of phospho-VEGF-R2 and phospho-SHP1 were also evaluated in BREC treated with or without 12-HETE by WB. In vivo studies were performed using streptozotocin-induced diabetic mice treated with or without 12/15-LOX inhibitor (baicalein, 75 mg/kg in drinking water). LC/MS was used to measure HETE production. Multiplex Immunoassay was used to measure adhesion molecules (ICAM-1 and VCAM-1) and IL6. DHE and immunofluorescence were used to measure ROS generation and NOX2 expression respectively in retinal sections from different groups.

Results: : We noticed significant increase in FITC-dextran flux across the BREC and reduction in the TER by 12- and 15- HETEs compared to the control. The change in REC permeability was time and dose dependent. This was associated with marked increases in ROS generation, NOX2 expression and levels of p-VEGF-R2 expression and reduction in the levels of p-SHP1 (P<0.05). In vivo studies demonstrated marked reduction in retinal HETEs, adhesion molecules (ICAM-1 and VCAM-1), IL6, ROS generation and NOX2 expression in diabetic mice treated with baicalein (P<0.05).

Conclusions: : Our findings suggest that 12/15-LOX contributes to early inflammatory response and breakdown of BRB in diabetic retina via NADPH oxidase dependent mechanism. This may involve inactivation of protein tyrosin phosphatase (SHP1) and activation of VEGF-R2.

Keywords: diabetic retinopathy • eicosanoids • oxidation/oxidative or free radical damage 
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