title: Targeting of 12/15-Lipoxygenase in retinal endothelial cells, but not in monocytes/macrophages, attenuates high glucose-induced retinal leukostasis.
abstract: Our previous studies have established a role for 12/15-lipoxygenase (LO) in mediating the inflammatory response in diabetic retinopathy (DR). However, the extent at which the local or systemic induction of 12/15-LO activity involved is unclear. Thus, the current study aimed to characterize the relative contribution of retinal endothelial versus monocytic/macrophagic 12/15-LO to inflammatory responses in DR.
  
  
  
  We first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of streptozotocin (STZ)-induced diabetic mice using liquid chromatography coupled with mass-spectrometry (LC-MS) to evaluate changes in circulating 12/15-LO activity. This was followed by comparing the in vitro mouse endothelium-leukocytes interaction between leukocytes isolated from 12/15-LO knockout (KO) versus those isolated from wild type (WT) mice using the myeloperoxidase (MPO) assay. Finally, we examined the effects of knocking down or inhibiting endothelial 12/15-LO on diabetes-induced endothelial cell activation and ICAM-1 expression.
  
  
  
  Analysis of plasma bioactive lipids' heat map revealed that the activity of circulating 12/15-LO was not altered by diabetes as evident by no significant changes in the plasma levels of major metabolites derived from 12/15-lipoxygenation of different PUFAs, including linoleic acid (13-HODE), arachidonic acid (12- and 15- HETEs), eicosapentaenoic acid (12- and 15- HEPEs), or docosahexaenoic acid (17-HDoHE). Moreover, leukocytes from 12/15-LO KO mice displayed a similar increase in adhesion to high glucose (HG)-activated endothelial cells as do leukocytes from WT mice. Furthermore, abundant proteins of 12-LO and 15-LO were detected in human retinal endothelial cells (HRECs), while it was undetected (15-LO) or hardly detectable (12-LO) in human monocyte-like U937 cells. Inhibition or knock down of endothelial 12/15-LO in HRECs blocked HG-induced expression of ICAM-1, a well-known identified important molecule for leukocyte adhesion in DR.
  
  
  
  Our data support that endothelial, rather than monocytic/macrophagic, 12/15-LO has a critical role in hyperglycemia-induced ICAM-1 expression, leukocyte adhesion, and subsequent local retinal barrier dysfunction. This may facilitate the development of more precisely targeted treatment strategies for DR.
authors.affiliation: Department of Oral Biology and Anatomy, Dental College of Georgia, Augusta University, Augusta, GA, USA; Department of Ophthalmology and Culver Vision Discovery Institute, Medical College of Georgia (MCG), Augusta University, Augusta, GA, USA; Department of Biochemistry and Clinical Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt.
authors.forename: Ahmed S
authors.initials: AS
authors.lastname: Ibrahim
authors.affiliation: Department of Oral Biology and Anatomy, Dental College of Georgia, Augusta University, Augusta, GA, USA; Department of Ophthalmology and Culver Vision Discovery Institute, Medical College of Georgia (MCG), Augusta University, Augusta, GA, USA.
authors.forename: Heba
authors.initials: H
authors.lastname: Saleh
authors.affiliation: Department of Oral Biology and Anatomy, Dental College of Georgia, Augusta University, Augusta, GA, USA; Department of Anatomy, Faculty of Medicine, Mansoura University, Mansoura, Egypt.
authors.forename: Mohamed
authors.initials: M
authors.lastname: El-Shafey
authors.affiliation: Department of Oral Biology and Anatomy, Dental College of Georgia, Augusta University, Augusta, GA, USA; Oral Medicine and Surgery Department, Oral and Dental Research Division, National Research Centre, USA.
authors.forename: Khaled A
authors.initials: KA
authors.lastname: Hussein
authors.affiliation: Department of Oral Biology and Anatomy, Dental College of Georgia, Augusta University, Augusta, GA, USA; Department of Anatomy, Faculty of Medicine, Mansoura University, Mansoura, Egypt; Cellular Biology and Anatomy, MCG, Augusta University, Augusta, GA, USA.
authors.forename: Khaled
authors.initials: K
authors.lastname: El-Masry
authors.affiliation: Department of Oral Biology and Anatomy, Dental College of Georgia, Augusta University, Augusta, GA, USA; Section of Plastic Surgery, Department of Surgery, Augusta University, Augusta, GA, USA.
authors.forename: Babak
authors.initials: B
authors.lastname: Baban
authors.affiliation: Department of Ophthalmology and Visual Sciences, and Biomedical Engineering, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
authors.forename: Nader
authors.initials: N
authors.lastname: Sheibani
authors.affiliation: Department of Physiology, MCG, Augusta University, Augusta, GA, USA.
authors.forename: Mong-Heng
authors.initials: MH
authors.lastname: Wang
authors.affiliation: Department of Oral Biology and Anatomy, Dental College of Georgia, Augusta University, Augusta, GA, USA; Department of Ophthalmology and Culver Vision Discovery Institute, Medical College of Georgia (MCG), Augusta University, Augusta, GA, USA; Cellular Biology and Anatomy, MCG, Augusta University, Augusta, GA, USA.
authors.forename: Amany
authors.initials: A
authors.lastname: Tawfik
authors.affiliation: Department of Oral Biology and Anatomy, Dental College of Georgia, Augusta University, Augusta, GA, USA; Department of Ophthalmology and Culver Vision Discovery Institute, Medical College of Georgia (MCG), Augusta University, Augusta, GA, USA; Department of Anatomy, Faculty of Medicine, Mansoura University, Mansoura, Egypt; Cellular Biology and Anatomy, MCG, Augusta University, Augusta, GA, USA. Electronic address: malshabrawey@augusta.edu.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Al-Shabrawey
mesh_terms: D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D001093:Arachidonate 15-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D002448:Cell Adhesion / Q000235:genetics; D003930:Diabetic Retinopathy / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D042783:Endothelial Cells / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis / Q000235:genetics; D018921:Leukostasis / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D008264:Macrophages / Q000201:enzymology* / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D009000:Monocytes / Q000201:enzymology* / Q000473:pathology; D012160:Retina / Q000201:enzymology* / Q000473:pathology; D020298:U937 Cells
chemical_list: C475064:12-15-lipoxygenase; C090509:Icam1 protein, mouse; D018799:Intercellular Adhesion Molecule-1; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase
keywords: 12-HETE; 12/15-Lipoxygenase; 15-HETE; Bioactive lipids; Blood retinal barrier; Diabetic retinopathy; Eicosanoids; ICAM-1; Leukostasis; Permeability