Aims/hypothesisIntra-retinal extravasation and modification of LDL have been implicated in diabetic retinopathy: autophagy may mediate these effects.MethodsImmunohistochemistry 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.ResultsIntra-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.ConclusionsAutophagy 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-016-4058-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

PurposeLimited mechanistic understanding of diabetic retinopathy (DR) has hindered therapeutic advances. Berberine, an isoquinolone alkaloid, has shown favorable effects on glucose and lipid metabolism in animal and human studies, but effects on DR are unknown. We previously demonstrated intraretinal extravasation and modification of LDL in human diabetes, and toxicity of modified LDL to human retinal Müller cells. We now explore pathogenic effects of modified LDL on Müller cells, and the efficacy of berberine in mitigating this cytotoxicity.MethodsConfluent human Müller cells were exposed to in vitro–modified ‘highly oxidized, glycated (HOG-) LDL versus native-LDL (N-LDL; 200 mg protein/L) for 6 or 24 hours, with/without pretreatment with berberine (5 μM, 1 hour) and/or the adenosine monophosphate (AMP)-activated protein kinase (AMPK) inhibitor, Compound C (5 μM, 1 hour). Using techniques including Western blots, reactive oxygen species (ROS) detection assay, and quantitative real-time PCR, the following outcomes were assessed: cell viability (CCK-8 assay), autophagy (LC3, Beclin-1, ATG-5), apoptosis (cleaved caspase 3, cleaved poly-ADP ribose polymerase), oxidative stress (ROS, nuclear factor erythroid 2-related factor 2, glutathione peroxidase 1, NADPH oxidase 4), angiogenesis (VEGF, pigment epithelium-derived factor), inflammation (inducible nitric oxide synthase, intercellular adhesion molecule 1, IL-6, IL-8, TNF-α), and glial cell activation (glial fibrillary acidic protein).ResultsNative-LDL had no effect on cultured human Müller cells, but HOG-LDL exhibited marked toxicity, significantly decreasing viability and inducing autophagy, apoptosis, oxidative stress, expression of angiogenic factors, inflammation, and glial cell activation. Berberine attenuated all the effects of HOG-LDL (all P < 0.05), and its effects were mitigated by AMPK inhibition (P < 0.05).ConclusionsBerberine inhibits modified LDL-induced Müller cell injury by activating the AMPK pathway, and merits further study as an agent for preventing and/or treating DR.

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Purpose: There is a lack of treatment for early diabetic retinopathy (DR), including blood-retina barrier (BRB) breakdown. The robust clinical benefit of fenofibrate in DR provides an opportunity to explore disease mechanisms and therapeutic targets. We have previously found that modified lipoproteins contribute to DR and that fenofibrate protects the inner BRB. We now investigate (1) whether modified lipoproteins elicit outer BRB injury and (2) whether fenofibrate may alleviate such damage. Methods: Human retinal pigment epithelium ARPE-19 cells were cultured in semipermeable transwells to establish a monolayer barrier and then exposed to heavily oxidized, glycated low-density lipoprotein (HOG-LDL, 25-300 mg/L, up to 24 h) versus native (N)-LDL. Transepithelial electric resistance (TEER) and FITCdextran permeability were measured. The effects of fenofibrate, its active metabolite fenofibric acid, and other peroxisome proliferator-activated receptor (PPARa) agonists (gemfibrozil, bezafibrate, and WY14643) were evaluated, with and without the PPARa antagonist GW6471 or the adenosine monophosphate-activated protein kinase (AMPK) inhibitor Compound C. Results: HOG-LDL induced concentration-and time-dependent barrier impairment, decreasing TEER and increasing dextran leakage, effects that were amplified by high glucose. Fenofibric acid, but not fenofibrate, gemfibrozil, bezafibrate, or WY14643, attenuated barrier impairment. This effect was reversed significantly by Compound C, but not by GW6471. Conclusions: Modified lipoproteins elicited outer BRB injury in an experimental model, which was reduced by fenofibric acid through a PPARa-independent, AMPK-mediated mechanism. These findings suggest a protective role of fenofibric acid on the outer BRB in diabetic retina.