Aims/hypothesis In previous studies we have shown that extravasated, modified LDL is associated with pericyte loss, an early feature of diabetic retinopathy (DR). Here we sought to determine detailed mechanisms of this LDL-induced pericyte loss. Methods Human retinal capillary pericytes (HRCP) were exposed to ‘highly-oxidised glycated’ LDL (HOG-LDL) (a model of extravasated and modified LDL) and to 4-hydroxynonenal or 7-ketocholesterol (components of oxidised LDL), or to native LDL for 1 to 24 h with or without 1 h of pretreatment with inhibitors of the following: (1) the scavenger receptor (polyinosinic acid); (2) oxidative stress (N-acetyl cysteine); (3) endoplasmic reticulum (ER) stress (4-phenyl butyric acid); and (4) mitochondrial dysfunction (cyclosporin A). Oxidative stress, ER stress, mitochondrial dysfunction, apoptosis and autophagy were assessed using techniques including western blotting, immunofluorescence, RT-PCR, flow cytometry and TUNEL assay. To assess the relevance of the results in vivo, immunohistochemistry was used to detect the ER stress chaperon, 78 kDa glucose-regulated protein, and the ER sensor, activating transcription factor 6, in retinas from a mouse model of DR that mimics exposure of the retina to elevated glucose and elevated LDL levels, and in retinas from human participants with and without diabetes and DR. Results Compared with native LDL, HOG-LDL activated oxidative and ER stress in HRCP, resulting in mitochondrial dysfunction, apoptosis and autophagy. In a mouse model of diabetes and hyperlipidaemia (vs mouse models of either condition alone), retinal ER stress was enhanced. ER stress was also enhanced in diabetic human retina and correlated with the severity of DR. Conclusions/interpretation Cell culture, animal, and human data suggest that oxidative stress and ER stress are induced by modified LDL, and are implicated in pericyte loss in DR.

Dietary flavonoid intake, especially berry flavonoids, has been associated with reduced risks of cardiovascular disease (CVD) in large prospective cohorts. Few clinical studies have examined the effects of dietary berries on CVD risk factors. We examined the hypothesis that freeze-dried strawberries (FDS) improve lipid and lipoprotein profiles and lower biomarkers of inflammation and lipid oxidation in adults with abdominal adiposity and elevated serum lipids. In a randomized dose-response controlled trial, 60 volunteers [5 men and 55 women; aged 49 ± 10 y; BMI: 36 ± 5 kg/m(2) (means ± SDs)] were assigned to consume 1 of the following 4 beverages for 12 wk: 1) low-dose FDS (LD-FDS; 25 g/d); 2) low-dose control (LD-C); 3) high-dose FDS (HD-FDS; 50 g/d); and 4) high-dose control (HD-C). Control beverages were matched for calories and total fiber. Blood draws, anthropometrics, blood pressure, and dietary data were collected at screening (0 wk) and after 12-wk intervention. Dose-response analyses revealed significantly greater decreases in serum total and LDL cholesterol and nuclear magnetic resonance (NMR)-derived small LDL particle concentration in HD-FDS [33 ± 6 mg/dL, 28 ± 7 mg/dL, and 301 ± 78 nmol/L, respectively (means ± SEMs)] vs. LD-FDS (-3 ± 11 mg/dL, -3 ± 9 mg/dL, and -28 ± 124 nmol/L, respectively) over 12 wk (0-12 wk; all P < 0.05). Compared with controls, only the decreases in total and LDL cholesterol in HD-FDS remained significant vs. HD-C (0.7 ± 12 and 1.4 ± 9 mg/dL, respectively) over 12 wk (0-12 wk; all P < 0.05). Both doses of strawberries showed a similar decrease in serum malondialdehyde at 12 wk (LD-FDS: 1.3 ± 0.2 μmol/L; HD-FDS: 1.2 ± 0.1 μmol/L) vs. controls (LD-C: 2.1 ± 0.2 μmol/L; HD-C: 2.3 ± 0.2 μmol/L) (P < 0.05). In general, strawberry intervention did not affect any measures of adiposity, blood pressure, glycemia, and serum concentrations of HDL cholesterol and triglycerides, C-reactive protein, and adhesion molecules. Thus, HD-FDS exerted greater effects in lowering serum total and LDL cholesterol and NMR-derived small LDL particles vs. LD-FDS in the 12-wk study. These findings warrant additional investigation in larger trials. This trial was registered at clinicaltrials.gov as NCT01883401.

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.

Aims/hypothesis Blood–retina barrier leakage in diabetes results in extravasation of plasma lipoproteins. Intra-retinal modified LDL have been implicated in diabetic retinopathy (DR), but their effects on retinal pigment epithelial (RPE) cells and the added effects of extravasated modified HDL are unknown. Methods In human retinas from individuals with and without diabetes and DR, immunohistochemistry was used to detect ApoB, ApoA1 and endoplasmic reticulum (ER) stress markers. In cell culture, human RPE cells were treated with native LDL (N-LDL) or heavily-oxidised glycated LDL (HOG-LDL) with or without pretreatment with native HDL (N-HDL) or heavily-oxidised glycated HDL (HOG-HDL). Cell viability, oxidative stress, ER stress, apoptosis and autophagy were assessed by Cell Counting Kit-8 assay, dichlorofluorescein assay, western blotting, immunofluorescence and TUNEL assay. In separate experiments, RPE cells were treated with lipid oxidation products, 7-ketocholesterol (7-KC, 5–40 µmol/l) or 4-hydroxynonenal (4-HNE, 5–80 µmol/l), with or without pretreatment with N-HDL or HOG-HDL. Results ApoB, ApoA1 staining and RPE ER stress were increased in the presence of DR. HOG-LDL but not N-LDL significantly decreased RPE cell viability and increased reactive oxygen species generation, ER stress, apoptosis and autophagy. Similarly, 4-HNE and 7-KC decreased viability and induced ER stress. Pretreatment with N-HDL mitigated these effects, whereas HOG-HDL was less effective by most, but not all, measures. Conclusions/interpretation In DR, extravascular modified LDL may promote RPE injury through oxidative stress, ER stress, autophagy and apoptosis. N-HDL has protective effects, but HOG-HDL is less effective. Extravasation and modification of HDL may modulate the injurious effects of extravasated modified LDL on the retinal pigment epithelium.

Acute cocoa supplementation showed no clear overall benefit in T2D patients after a high-fat fast-food-style meal challenge. Although HDL cholesterol and insulin remained higher throughout the 6-h postprandial period, an overall decrease in large artery elasticity was found after cocoa consumption. This trial was registered at clinicaltrials.gov as NCT01886989.

PURPOSE. We previously showed that extravasated, modified LDL is implicated in pericyte loss in diabetic retinopathy (DR). Here, we investigate whether modified LDL induces apoptosis in retinal Müller glial cells. METHODS. Cultured human retinal Müller cells (MIO-M1) were treated with highly oxidized glycated LDL (HOG-LDL, 200 mg protein/L) or native LDL (N-LDL, 200 mg protein/L) for up to 24 hours with or without pretreatment with N-acetyl-cysteine (NAC, a blocker of oxidative stress) and 4-phenylbutyrate (4-PBA, a blocker of endoplasmic reticulum [ER] stress). Effects of HOG-LDL on cell viability, apoptosis, oxidative stress, and ER stress were assessed by cell viability, TUNEL, and Western blot assays. In separate experiments, Müller cells were treated with 7-ketocholesterol (7-KC, 5-20 lM) or 4-hydroxynonenal (4-HNE, 5-40 lM) for up to 24 hours. The same markers were measured.RESULTS. HOG-LDL induced apoptosis (decreased cell viability, increased TUNEL staining, increased expression of cleaved PARP, cleaved caspase-3, and BAX; decreased Bcl-2), oxidative stress (increased NOX4 and antioxidant enzymes, catalase, and superoxide dismutase 2), and ER stress (increased phospho-eIF2a, KDEL, ATF6, and CHOP). Pretreatment with NAC or 4-PBA partially attenuated apoptosis. In addition. NAC attenuated activation of ER stress. Similar to HOG-LDL, 7KC, and 4HNE also induced apoptosis, oxidative stress, and ER stress.CONCLUSIONS. Our data suggest that extravasated, modified lipoproteins may be implicated in apoptotic Müller cell death, acting at least partially via enhanced levels of oxidative and ER stresses. They support our main hypothesis that, in addition to hyperglycemia, extravasated and oxidized LDL is an important insult to the diabetic retina. (Invest Ophthalmol Vis Sci. 2012;53:4595-4604 In previous work, we proposed that in addition to hyperglycemia, extravasation of plasma lipoproteins through leaking blood retinal barriers (BRB) and their subsequent modification (glycation, oxidation) are important in the propagation of DR. 12-18 Several lines of evidence support this concept. Clinical studies indicate that dyslipidemia is associated with the severity of DR In particular, DR is positively associated with serum levels of LDL, apolipoprotein B (ApoB), and LDL particle concentration in type 1 diabetic patients. 13,[19][20][21] However, dyslipidemia in the absence of diabetes does not cause retinal injury, and we suggest that breakdown of the BRB is the critical factor. Using immunohistochemistry (for ApoB and oxidized LDL [ox-LDL]), we identified the presence of intraretinal modified LDL in type 2 diabetic patients who had not yet developed clinical DR, with larger amounts proportionate to disease severity in patients with clinical DR This staining initially surrounded the inner retinal capillaries. We also confirmed the absence of ApoB and ox-LDL in normal human retina. 16 In ex vivo studies, ox-LDL was associated with apoptotic figures in human diabetic retinas. 16 In more severe DR cases with proliferative D...

This article is available online at http://www.jlr.org Diabetic retinopathy (DR) remains a leading cause of vision loss in working-age adults. Early disease is characterized by vascular abnormalities, including pericyte loss, basement membrane thickening, microaneurysm formation, and capillary leakage ( 1, 2 ). Pericytes are critical to vascular integrity ( 3 ), and their loss is considered an initiating event in DR.Dyslipidemia has been implicated in DR, and associations between plasma lipoprotein profi les and disease severity have been observed in large cohort studies ( 4-6 ), but were not of suffi cient strength to defi ne risk for individuals. The term "dyslipidemia" may include qualitative as well as quantitative alterations in plasma lipoproteins. The qualitative abnormalities include modifi cation by oxidation, but oxidized LDL (ox-LDL) constitutes only a small fraction of total plasma LDL, ranging from 0.001% in healthy people to 5% in the presence of cardiovascular

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.