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

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 ?mol/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.

SUBMITTER: Elmasry K

PROVIDER: S-EPMC5878142 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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

Elmasry Khaled K Ibrahim Ahmed S AS Saleh Heba H Elsherbiny Nehal N Elshafey Sally S Hussein Khaled A KA Al-Shabrawey Mohamed M

Diabetologia 20180221 5

<h4>Aims/hypothesis</h4>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.<h4>Methods</h4>We used in vivo and in vitro approaches. For in vivo studies, experimental ...[more]

PMID: 29468369

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Project description:ObjectivesTo determine the risk factors for retinal microvascular impairment on optical coherence tomography angiography (OCT-A) in type 2 diabetic patients without clinical diabetic retinopathy (DR).MethodsThis retrospective and cross-sectional study enrolled 74 diabetic patients without clinically evident DR for the study group and 34 healthy subjects for the control group. OCT-A parameters were measured to determine vascular density (VD) and the foveal avascular zone (FAZ) size in the superficial and deep capillary plexuses (SCP/DCP) of the retina. Clinical data were collected on sex, age, diabetes duration, hemoglobin A1c (HbA1c), hypertension, dyslipidemia, low-density lipoprotein cholesterol (LDL-C), estimated glomerular filtration rate (eGFR) and smoking status. Multiple linear regression analyses were performed to represent the associated clinical variables with OCT-A parameters in diabetic patients.ResultsIn comparison between the study and control groups, the VD in the SCP and DCP were significantly lower in diabetic patients compared to the controls (P = 0.022 and 0.003, respectively). The FAZ size in the SCP and DCP were significantly greater in diabetic patients compared to the controls (P = 0.035 and <0.001, respectively). In age- and sex-adjusted multiple regression analyses for the diabetic patients, dyslipidemia and hypertension were negatively associated with SCP-VD (β = -0.357, P = 0.002; β = -0.239, P = 0.039, respectively). Current smoking was correlated with lower DCP-VD (β = -0.255, P = 0.043). Greater SCP-FAZ size was associated with dyslipidemia and greater LDL-C (β = 0.254, P = 0.013; β = 0.232, P = 0.029, respectively), and greater DCP-FAZ size, with lower eGFR and greater LDL-C (β = -0.355, P = 0.004; β = 0.235, P = 0.037, respectively).ConclusionsDiabetic patients without clinical DR showed lower VD and greater FAZ size in the SCP and DCP compared to healthy controls. In diabetic patients without clinical DR, dyslipidemia and/or high LDL-C were important risk factors for retinal microvascular impairment. Hypertension, current smoking and lower eGFR also contributed to microvascular impairment.

Project description:Importance:Quantitative measurements based on optical coherence tomographic angiography (OCTA) may have value in managing diabetic retinopathy (DR), but there is limited information on the ability of OCTA to distinguish eyes with DR. Objective:To evaluate the ability of measurements of retinal microvasculature using OCTA to distinguish healthy eyes from eyes with DR. Design, Setting, and Participants:In this prospective cross-sectional study, OCTA was used to examine the eyes of participants with type 2 diabetes with or without DR and the eyes of participants without diabetes from September 17, 2015, to April 6, 2016. Density maps based on superficial retinal layer (SRL) and deeper retinal layer (DRL) images were generated after a method to remove decorrelation tails was applied to the DRL images. Exposures:Both eyes of each participant were examined by means of a 3-mm OCTA scan and 7-field fundus photography using the Diabetic Retinopathy Severity Scale. Main Outcomes and Measures:Two measures were examined: perfusion density, based on the area of vessels, and vessel density, based on a map with vessels of 1-pixel width. The size of the foveal avascular zone was also calculated automatically, and so was the area under the receiver operating characteristic curve. Results:A total of 50 eyes from 26 participants with diabetes (10 women and 16 men; mean [SD] age, 64.9 [7.5] years) and 50 healthy eyes from 25 participants without diabetes (14 women and 11 men; mean [SD] age, 64.0 [7.1] years) were imaged. All participants were white. Vessel density measured in the SRL had the highest area under the receiver operating characteristic curve (0.893 [95% CI, 0.827-0.959]), compared with perfusion density in the SRL (0.794 [95% CI, 0.707-0.881]), foveal avascular zone area (0.472 [95% CI, 0.356-0.588]), and vessel density in the DRL (0.703 [95% CI, 0.601-0.805]). Vessel density in the SRL negatively correlated with best-corrected visual acuity (r?=?-0.28; P?=?.05) and severity of DR (r?=?-0.46; P?=?.001). Density metrics correlated with age. No correlation was detected between vascular density or foveal avascular zone metrics and hemoglobin A1C or duration of diabetes. Conclusions and Relevance:Vessel density measured by OCTA provides a quantitative metric of capillary closure that correlates with severity of DR and may allow staging, diagnosis, and monitoring that do not require subjective evaluation of fundus images.

Project description:BackgroundDiabetic retinopathy (DR) is a main complication of diabetes mellitus (DM). Recent studies have implicated microRNAs in human retinal microvascular endothelial cell (HRMEC) dysfunction. In this study, we aim to investigate the apoptotic promotion of miR-29b-3p by blocking SIRT1 in HRMEC for DR situation.MethodBlood samples were obtained from DR patients and controls. Dual-luciferase reporter assay using HEK-293T cells was performed to show the direct interaction of miR-29b-3p and the 3'UTR of SIRT1. HRMECs were exposed to 5.5 mmol/L of glucose (normal control), 5.5 mmol/L of glucose and 24.5 mmol/L of mannitol (osmotic pressure control), 30 mmol/L of glucose [hyperglycemia (HG)], 150 μmol/L of CoCl2 (hypoxia), and 30 mmol/L of glucose plus 150 μmol/L of CoCl2 (HG-CoCl2). To identify the regulating relationship between miR-29b-3p and SIRT1, HRMECs were transfected with miR-29b-3p mimics/inhibitors or their negative controls. SRT1720 was used as a SIRT1 agonist. Cell viability was assessed with the cell counting kit-8 (CCK-8) assay, and apoptotic cells were stained by one-step terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit. Gene and protein expression were assayed by quantitative real-time reverse transcriptase-PCR (RT-qPCR) and western blotting separately.ResultMiR-29b-3p was upregulated to 3.2-fold, and SIRT1 protein was downregulated to 65% in DR patients. Dual-luciferase reporter assay showed the direct interaction of miR-29b-3p and SIRT1. HRMECs were identified as >95% positive for CD31 and von Willebrand factor (vWF). MiR-29b-3p and Bax/Bcl-2 ratio was upregulated, whereas SIRT1 was downregulated in HRMECs in the HG-CoCl2 condition. Decreased cell viability and upregulated apoptosis were also found in HRMECs of the HG-CoCl2 condition. Upregulated miR-29b-3p decreased the expression of SIRT1 and increased the ratio of Bax/Bcl-2, whereas downregulated miR-29b-3p increased the expression of SIRT1 protein and downregulated the ratio of Bax/Bcl-2. SRT1720 rescued miR-29b-3p-induced HRMEC apoptosis via upregulating the expression of SIRT1 protein.ConclusionThe dysregulation of miR-29b-3p/SIRT1 is a potential mechanism of HRMEC apoptosis in DR. MiR-29b-3p/SIRT1 may be a potential therapeutic target for DR.

Dataset Information

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

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

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