Ahmed S Ibrahim1, Heba Saleh2, Mohamed El-Shafey3, Khaled A Hussein4, Khaled El-Masry5, Babak Baban6, Nader Sheibani7, Mong-Heng Wang8, Amany Tawfik9, Mohamed Al-Shabrawey10. 1. 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. 2. 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. 3. 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. 4. 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. 5. 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. 6. 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. 7. Department of Ophthalmology and Visual Sciences, and Biomedical Engineering, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA. 8. Department of Physiology, MCG, Augusta University, Augusta, GA, USA. 9. 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. 10. 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.
Abstract
AIMS: 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. MATERIALS & METHODS: 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. RESULTS: 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. CONCLUSION: 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.
AIMS: 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. MATERIALS & METHODS: We first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of streptozotocin (STZ)-induced diabeticmice 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. RESULTS: 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. CONCLUSION: 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.
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