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Diabetologia. 2016 Oct;59(10):2251-61. doi: 10.1007/s00125-016-4058-5. Epub 2016 Jul 30.

Survival or death: a dual role for autophagy in stress-induced pericyte loss in diabetic retinopathy.

Author information

1
Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK.
2
Department of Immunology, Harbin Medical University, Harbin, People's Republic of China.
3
Section of Endocrinology and Diabetes, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
4
Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC, USA.
5
Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK. t.lyons@qub.ac.uk.

Abstract

AIMS/HYPOTHESIS:

Intra-retinal extravasation and modification of LDL have been implicated in diabetic retinopathy: autophagy may mediate these effects.

METHODS:

Immunohistochemistry was used to detect autophagy marker LC3B in human and murine diabetic and non-diabetic retinas. Cultured human retinal capillary pericytes (HRCPs) were treated with in vitro-modified heavily-oxidised glycated LDL (HOG-LDL) vs native LDL (N-LDL) with or without autophagy modulators: green fluorescent protein-LC3 transfection; small interfering RNAs against Beclin-1, c-Jun NH(2)-terminal kinase (JNK) and C/EBP-homologous protein (CHOP); autophagy inhibitor 3-MA (5 mmol/l) and/or caspase inhibitor Z-VAD-fmk (100 μmol/l). Autophagy, cell viability, oxidative stress, endoplasmic reticulum stress, JNK activation, apoptosis and CHOP expression were assessed by western blots, CCK-8 assay and TUNEL assay. Finally, HOG-LDL vs N-LDL were injected intravitreally to STZ-induced diabetic vs control rats (yielding 50 and 200 mg protein/l intravitreal concentration) and, after 7 days, retinas were analysed for ER stress, autophagy and apoptosis.

RESULTS:

Intra-retinal autophagy (LC3B staining) was increased in diabetic vs non-diabetic humans and mice. In HRCPs, 50 mg/l HOG-LDL elicited autophagy without altering cell viability, and inhibition of autophagy decreased survival. At 100-200 mg/l, HOG-LDL caused significant cell death, and inhibition of either autophagy or apoptosis improved survival. Further, 25-200 mg/l HOG-LDL dose-dependently induced oxidative and ER stress. JNK activation was implicated in autophagy but not in apoptosis. In diabetic rat retina, 50 mg/l intravitreal HOG-LDL elicited autophagy and ER stress but not apoptosis; 200 mg/l elicited greater ER stress and apoptosis.

CONCLUSIONS:

Autophagy has a dual role in diabetic retinopathy: under mild stress (50 mg/l HOG-LDL) it is protective; under more severe stress (200 mg/l HOG-LDL) it promotes cell death.

KEYWORDS:

Apoptosis; Autophagy; Diabetic retinopathy; ER stress; LC3B; Modified LDL; Oxidative stress; Pericytes

PMID:
27475954
PMCID:
PMC5016562
DOI:
10.1007/s00125-016-4058-5
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

Compliance with ethical standards Funding This work was supported by the Oklahoma Center for the Advancement of Science and Technology (HR08-067), by a Research Grant from the American Diabetes Association (7-12-CT46), and by the National Institutes of Health (USA) COBRE Program of the National Center for Research Resources (P20 RR 024215). Duality of interest The authors declare that there is no duality of interest associated with this manuscript. Contribution statement DF, SY, MW, SMH, ARC and MD conducted experiments, researched data and wrote the manuscript. JYY and JC researched data and wrote the manuscript. TJL conceived and conducted the study, researched data and wrote the manuscript. TJL is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All the authors have approved publication.

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