Role of endoplasmic reticulum stress in 12/15-lipoxygenase-induced retinal microvascular dysfunction in a mouse model of diabetic retinopathy

Khaled Elmasry, Ahmed Salah Ibrahim, Heba Mohamed Saleh, Nehal Elsherbiny, Sally Elshafey, Khaled A. Hussein, Mohamed Al-Shabrawey

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Aims/hypothesis: Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy. Methods: We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo−/−); ER stress was then evaluated after 12–14 weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2−/− mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1 μmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca2+) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. Results: Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leucocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO-derived metabolites both in vitro and in vivo. We also found that 15-HETE increases the intracellular calcium in HRECs. Conclusions/interpretation: ER stress contributes to 12/15-LO-induced retinal inflammation in diabetic retinopathy via activation of NADPH oxidase and VEGFR2. Perturbation of calcium homeostasis in the retina might also play a role in linking 12/15-LO to retinal ER stress and subsequent microvascular dysfunction in diabetic retinopathy.

Original languageEnglish (US)
Pages (from-to)1220-1232
Number of pages13
JournalDiabetologia
Volume61
Issue number5
DOIs
StatePublished - May 1 2018

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Endoplasmic Reticulum Stress
Diabetic Retinopathy
Hydroxyeicosatetraenoic Acids
NADPH Oxidase
Endothelial Cells
Calcium
Retina
Activating Transcription Factor 6
12-15-lipoxygenase
eIF-2 Kinase
Protein Disulfide-Isomerases
Vascular Endothelial Growth Factor Receptor-2
Intravitreal Injections
Immunoprecipitation
Knockout Mice
Confocal Microscopy
Endoplasmic Reticulum
Catalytic Domain
Leukocytes
Homeostasis

Keywords

  • 12-HETE
  • 12/15-Lipoxygenase
  • 15-HETE
  • Bioactive lipids
  • Calcium
  • Diabetic retinopathy
  • ER stress
  • Eicosanoids
  • NADPH oxidase
  • VEGFR2

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism

Cite this

Role of endoplasmic reticulum stress in 12/15-lipoxygenase-induced retinal microvascular dysfunction in a mouse model of diabetic retinopathy. / Elmasry, Khaled; Ibrahim, Ahmed Salah; Saleh, Heba Mohamed; Elsherbiny, Nehal; Elshafey, Sally; Hussein, Khaled A.; Al-Shabrawey, Mohamed.

In: Diabetologia, Vol. 61, No. 5, 01.05.2018, p. 1220-1232.

Research output: Contribution to journalArticle

Elmasry, Khaled ; Ibrahim, Ahmed Salah ; Saleh, Heba Mohamed ; Elsherbiny, Nehal ; Elshafey, Sally ; Hussein, Khaled A. ; Al-Shabrawey, Mohamed. / Role of endoplasmic reticulum stress in 12/15-lipoxygenase-induced retinal microvascular dysfunction in a mouse model of diabetic retinopathy. In: Diabetologia. 2018 ; Vol. 61, No. 5. pp. 1220-1232.
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abstract = "Aims/hypothesis: Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy. Methods: We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo−/−); ER stress was then evaluated after 12–14 weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2−/− mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1 μmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca2+) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. Results: Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leucocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO-derived metabolites both in vitro and in vivo. We also found that 15-HETE increases the intracellular calcium in HRECs. Conclusions/interpretation: ER stress contributes to 12/15-LO-induced retinal inflammation in diabetic retinopathy via activation of NADPH oxidase and VEGFR2. Perturbation of calcium homeostasis in the retina might also play a role in linking 12/15-LO to retinal ER stress and subsequent microvascular dysfunction in diabetic retinopathy.",
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AU - Ibrahim, Ahmed Salah

AU - Saleh, Heba Mohamed

AU - Elsherbiny, Nehal

AU - Elshafey, Sally

AU - Hussein, Khaled A.

AU - Al-Shabrawey, Mohamed

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N2 - Aims/hypothesis: Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy. Methods: We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo−/−); ER stress was then evaluated after 12–14 weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2−/− mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1 μmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca2+) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. Results: Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leucocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO-derived metabolites both in vitro and in vivo. We also found that 15-HETE increases the intracellular calcium in HRECs. Conclusions/interpretation: ER stress contributes to 12/15-LO-induced retinal inflammation in diabetic retinopathy via activation of NADPH oxidase and VEGFR2. Perturbation of calcium homeostasis in the retina might also play a role in linking 12/15-LO to retinal ER stress and subsequent microvascular dysfunction in diabetic retinopathy.

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KW - Diabetic retinopathy

KW - ER stress

KW - Eicosanoids

KW - NADPH oxidase

KW - VEGFR2

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