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

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.

‘Teaching corner’: Management of Diabetic Retinopathy

Sub-Saharan Africa faces an epidemic of diabetes. Visual loss from diabetic retinopathy (DR) is both preventable and treatable. This article reviews the epidemiology and clinical features of DR and current evidencebased interventions in three areas: primary prevention of retinopathy by optimum medical management, early detection of pre-symptomatic disease and management of established retinopathy to prevent or mitigate visual loss. There are significant challenges to DR care in resource poor environments. Appropriate provision of effective interventions by health services can reduce social and economic costs associated with patient care.

Prevalence of diabetic retinopathy in type II diabetic patients in a health facility in Karachi, Pakistan

Purpose: To study the prevalence of diabetic retinopathy in known diabetic patients attending the diabetes outpatient department (OPD) of Sind Government Hospital (SGH), New Karachi Township (NKT), Pakistan. Methods: A cross-sectional observational study was carried out at the diabetic OPD of SGH, NKT over the period of 17 months from March 2013 to August 2014. The selected patients were interviewed based on a questionnaire; laboratory investigations were performed and examination of the eye was conducted by a specialist ophthalmologist. One hundred and fifty four (154) subjects out of 305 patients contacted fully completed the study. Stratification of the data on gender basis was done, after which one-way ANOVA, χ2 test of correlation, binary logistic regression and relative risk analyses were carried out using SPSS-20. Results: It was found that 66 % men of normal weight (χ2 = 4.667, p < 0.05) and 60.7 % overweight women (χ2 = 5.143, p < 0.05) were more likely to present with diabetic retinopathy (DR). Prevalence of DR in this target population was 42.86 % (N = 66). Background DR (56 %) and maculopathy (23 %) were more prevalent than advanced conditions of the disease. There was no gender-based preponderance for the presentation of DR (χ2 = 0.663; p > 0.05), nor was this seen in different ethnic groups. Conclusion: DR is prevalent in the target population and, therefore, emphasis should be on the education of the local population of New Karachi Township on how to attain euglycemic state with regular medication, diet and exercise to avoid development and progress of DR.

Management and outcome of cholesterol embolus identified in a diabetic retinopathy screening program

Purpose. We examined the incidence, management, and outcomes of patients known to be at high cardiovascular risk, and to assess whether specialist referral to an ophthalmic medical clinic is worthwhile. Methods. Patients in the East Birmingham area with cholesterolembolus who were identified on digital diabetic retinopathy screening over a 3-year period were referred to a specialist ophthalmic medicine clinic within Heart of England NHS Trust for management and investigation. Results. A total of 33 patients were referred for clinical management.(male:female = 22:11, mean age 72 years). A total of 28 patients were known to be receiving medication: 14 anti hypertensive therapy(42%), 19 aspirin (59%), and 21 statin (64%). A total of 18 patients had known cardiovascular disease, 10 of whom had received carotid stenting or coronary artery bypass surgery. Ten patients diagnosed with embolus required and consented to carotid Doppler studies. Six patients were confirmed with significant carotid stenosis and 2 (6%)of these patients required carotid endarterectomy surgery. Overall, 4patients died, a mortality rate of 12% over 3 years. Conclusions. Annual diabetic retinopathy screening provide sopportunistic identification of asymptomatic cholesterol emboli and provides an opportunity for review of medical management in the high-risk patient group with appropriate identification and referral for carotid stenosis surgery. A total of 11 patients were identified with sub optimal cardiovascular risk management: e.g., statin use.

Prevalence of retinopathy in Caucasian type 2 diabetic patients from the South of Brazil and relationship with clinical and metabolic factors

Diabetic retinopathy (DR) is a sight-threatening chronic complication of diabetes mellitus and is the leading cause of acquired blindness in adults. In this cross-sectional study, we investigated the prevalence of and the factors associated with DR in an analysis of 210 consecutive and unrelated Brazilian Caucasians with type 2 diabetes mellitus. Retinopathy was evaluated by ophthalmoscopy and/or biomicroscopy through dilated pupils. The relationship between clinical and metabolic variables and the presence of DR was assessed by logistic regression analysis. DR was detected in 99 of the 210 patients (47%). In the univariate logistic regression analyses, male sex, duration of diabetes, body mass index, glycated hemoglobin, C-peptide, LDL cholesterol, smoking, and albumin excretion rate were found to be associated with the presence of DR. However, the multiple logistic regression analysis showed that only duration of diabetes (odds ratio (OR) = 1.15, 95% CI = 1.09-1.22; P < 0.001), glycated hemoglobin (OR = 1.21, 95% CI = 1.01-1.46; P = 0.047) and albumin excretion rate >100 µg/min (OR = 12.72, 95% CI = 3.89-41.56; P < 0.001) were independently associated with DR. Although DR was found to be frequent among Brazilian type 2 diabetic patients, its prevalence was within the range observed in other Caucasian populations. Our findings emphasize the need for good glycemic control in order to prevent or delay the onset of DR, since the most well-known risk factors for the development of this complication in type 2 diabetes mellitus, such as duration of diabetes, glycated hemoglobin and albumin excretion rate were independently related to DR.

Higher prevalence of retinopathy in diabetic patients of South Asian ethnicity compared with white Europeans in the community:a cross-sectional study

OBJECTIVE—The purpose of this study was to compare prevalence and risk factors for diabetic retinopathy among U.K. residents of South Asian or white European ethnicity. RESEARCH DESIGN AND METHODS—This was a community-based cross-sectional study involving 10 general practices; 1,035 patients with type 2 diabetes were studied: 421 of South Asian and 614 of white European ethnicity. Diabetic retinopathy, sight-threatening retinopathy, maculopathy, and previous laser photocoagulation therapy were assessed after grading of retinal photographs. Data were collected on risk factors including age, duration, and treatment of diabetes, blood pressures, serum total cholesterol, and A1C. RESULTS—Patients of South Asian ethnicity had significantly higher systolic (144 vs. 137 mmHg, P < 0.0001) and diastolic (84 vs. 74 mmHg, P < 0.0001) blood pressure, A1C (7.9 vs. 7.5%, P < 0.0001), and total cholesterol (4.5 vs. 4.2 mmol/l, P < 0.0001). Diabetic retinopathy was detected in 414 (40%) patients (189 South Asian [45%] versus 225 white European [37%]; P = 0.0078). Sight-threatening retinopathy was detected in 142 (14%) patients (68 South Asian [16%] versus 74 white European [12%]; P = 0.0597). After adjustment for confounders, there were significantly elevated risks of any retinopathy and maculopathy for South Asian versus white European patients. CONCLUSIONS—Patients of South Asian ethnicity had a significantly higher prevalence of diabetic retinopathy and maculopathy, with significantly elevated systolic and diastolic blood pressure, A1C, and total cholesterol; lower attained age; and younger age at diagnosis. Earlier onset of disease and higher levels of modifiable risk factors make early detection of diabetes, annual referral for retinal screening, and intensive risk factor control key elements in addressing this health inequality.

Diagnosis of retinopathy in children younger than 12 years of age:implications for the diabetic eye screening guidelines in the UK

Aim: To assess whether the current starting age of 12 is suitable for diabetic retinopathy (DR) screening and whether diabetes duration should be taken into account when deciding at what age to start screening patients. Materials and methods: A retrospective analysis of 143 patients aged 12 years or younger who attended diabetic eye screening for the first time in the Birmingham, Solihull and Black Country Diabetic Eye Screening Programme was performed. Results: The mean age of the patients was 10.7 (7-12) years with 73 out of 143 aged below 12 years and 70 were 12 years of age. 98% had type 1 diabetes and mean diabetes duration was 5 (1 month-11 years) years. For those younger than 12 years, 7/73 (9.6%) had background DR (BDR), of these mean diabetes duration was 7 years (6-8). The youngest patient to present with DR was aged 8 years. In those aged 12 years, 5/70 (7.1%) had BDR; of these mean diabetes duration was 8 years (6-11). No patient developed DR before 6 years duration in either group. Conclusions: The results show that no patient younger than the age of 12 had sight-threatening DR (STDR), but BDR was identified. Based on the current mission statement of the Diabetic Eye Screening Programme to identify STDR, 12 years of age is confirmed as the right age to start screening, but if it is important to diabetic management to identify first development of DR, then screening should begin after 6 years of diabetes diagnosis.

Endocytosis Of Tight Junctions Caveolin Nitrosylation Dependent Is Improved By Cocoa Via Opioid Receptor On Rpe Cells In Diabetic Conditions.

Retinal pigment epithelium cells, along with tight junction (TJ) proteins, constitute the outer blood retinal barrier (BRB). Contradictory findings suggest a role for the outer BRB in the pathogenesis of diabetic retinopathy (DR). The aim of this study was to investigate whether the mechanisms involved in these alterations are sensitive to nitrosative stress, and if cocoa or epicatechin (EC) protects from this damage under diabetic (DM) milieu conditions. Cells of a human RPE line (ARPE-19) were exposed to high-glucose (HG) conditions for 24 hours in the presence or absence of cocoa powder containing 0.5% or 60.5% polyphenol (low-polyphenol cocoa [LPC] and high-polyphenol cocoa [HPC], respectively). Exposure to HG decreased claudin-1 and occludin TJ expressions and increased extracellular matrix accumulation (ECM), whereas levels of TNF-α and inducible nitric oxide synthase (iNOS) were upregulated, accompanied by increased nitric oxide levels. This nitrosative stress resulted in S-nitrosylation of caveolin-1 (CAV-1), which in turn increased CAV-1 traffic and its interactions with claudin-1 and occludin. This cascade was inhibited by treatment with HPC or EC through δ-opioid receptor (DOR) binding and stimulation, thereby decreasing TNF-α-induced iNOS upregulation and CAV-1 endocytosis. The TJ functions were restored, leading to prevention of paracellular permeability, restoration of resistance of the ARPE-19 monolayer, and decreased ECM accumulation. The detrimental effects on TJs in ARPE-19 cells exposed to DM milieu occur through a CAV-1 S-nitrosylation-dependent endocytosis mechanism. High-polyphenol cocoa or EC exerts protective effects through DOR stimulation.

Polyphenol-enriched Cocoa Protects The Diabetic Retina From Glial Reaction Through The Sirtuin Pathway.

Cocoa is rich in flavonoids, which are potent antioxidants with established benefits for cardiovascular health but unproven effects on neurodegeneration. Sirtuins (SIRTs), which make up a family of deacetylases, are thought to be sensitive to oxidation. In this study, the possible protective effects of cocoa in the diabetic retina were assessed. Rat Müller cells (rMCs) exposed to normal or high glucose (HG) or H2O2 were submitted to cocoa treatment in the presence or absence of SIRT-1 inhibitor and small interfering RNA The experimental animal study was conducted in streptozotocin-induced diabetic rats randomized to receive low-, intermediate-, or high-polyphenol cocoa treatments via daily gavage for 16 weeks (i.e., 0.12, 2.9 or 22.9 mg/kg/day of polyphenols). The rMCs exposed to HG or H2O2 exhibited increased glial fibrillary acidic protein (GFAP) and acetyl-RelA/p65 and decreased SIRT1 activity/expression. These effects were cancelled out by cocoa, which decreased reactive oxygen species production and PARP-1 activity, augmented the intracellular pool of NAD(+), and improved SIRT1 activity. The rat diabetic retinas displayed the early markers of retinopathy accompanied by markedly impaired electroretinogram. The presence of diabetes activated PARP-1 and lowered NAD(+) levels, resulting in SIRT1 impairment. This augmented acetyl RelA/p65 had the effect of up-regulated GFAP. Oral administration of polyphenol cocoa restored the above alterations in a dose-dependent manner. This study reveals that cocoa enriched with polyphenol improves the retinal SIRT-1 pathway, thereby protecting the retina from diabetic milieu insult.

Comparison of full-field electroretinogram in diabetic and non diabetic dogs with cataracts

Being the commonest ocular disorder, dense cataracts disable fundoscopic examination and the diagnosis of retinal disorders, which dogs may be predisposed. The aim of this study was to compare the electroretinographic responses recorded according to the International Society for Clinical Electrophysiology of Vision human protocol to evaluate retinal function of diabetic and non diabetic dogs, both presenting mature or hypermature cataracts. Full-field electroretinogram was recorded from 66 dogs, with ages varying from 6 to 15 years old allocated into two groups: (1) CG, non diabetic cataractous dogs, and (2) DG, diabetic cataractous dogs. Mean peak-to-peak amplitude (microvolts) and b-wave implicit time (milliseconds) were determined for each of the five standard full-field ERG responses (rod response, maximal response, oscillatory potentials, single-flash cone response and 30 Hz flicker). Comparing CG to DG, ERGs recorded from diabetic dogs presented lower amplitude and prolonged b-wave implicit time in all ERG responses. Prolonged b-wave implicit time was statistically significant (p< 0.05) at 30 Hz flicker (24.0 ms versus 22.4 ms). These data suggests full-field ERG is capable to record sensible alterations, such as flicker's implicit time, being useful to investigate retinal dysfunction in diabetic dogs.

Expression and cellular localization of microRNA-29b and RAX, an activator of the RNA-dependent protein kinase (PKR), in the retina of streptozotocin-induced diabetic rats

Purpose: The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats. Methods: Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis. Results: We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b. Conclusions: Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.

Intravitreal Bevacizumab for Diabetic Macular Edema Associated With Severe Capillary Loss: One-Year Results of a Pilot Study

PURPOSE: To evaluate the effects of intravitreal bevacizumab in patients with diabetic macular edema (DME) associated with severe capillary loss. DESIGN: Multicenter, open-label, nonrandomized study. METHODS: SETTING: Two tertiary ophthalmic referral centers in Brazil. STUDY POPULATION: Ten consecutive patients with DME and ""severe"" capillary loss. OBSERVATION PROCEDURES: Intravitreal injection(s) of bevacizumab (1.5 mg). Standardized ophthalmic evaluation was performed at baseline and at weeks 8, 16, 24, and 54. MAIN OUTCOME MEASURES: Changes in best-corrected visual acuity (BCVA) and in optical coherence tomography variables (central macular thickness [CMT] and total macular volume [TMV]). RESULTS: Significant changes in BCVA and in CMT/TMV were noted throughout the study (P<.001, P=.009, and P<.001, respectively). The mean logarithm of the minimal angle of resolution Early Treatment Diabetic Retinopathy Study BCVA was 0.786 (similar to 20/125(+1)) at baseline, 0.646 (similar to 20/80(-2)) at week 8, 0.580 (20/80(+1)) at week 16, 0.574 (similar to 20/80(+1)) at week 24, and 0.558 (similar to 20/80(+2)) at week 54. Compared with baseline, a significant change in BCVA was noted at all follow-up visits (P <=.008). The mean CMT/TMV values were, respectively, 472.6/10.9 at baseline, 371.4/9.9 at week 8, 359.5/9.8 at week 16, 323.9/9-4 at week 24, and 274.6/8.7 at week 54. Compared with baseline, a significant change in both CMT and TMV was noted only at 24 and 54 weeks (P <=.007). At 54 weeks, fluorescein angiography demonstrated no change in the extent of macular capillary loss and reduced dye leakage as compared with baseline in all patients. CONCLUSIONS: Favorable changes in BCVA and in CMT/TMV observed throughout 1 year suggest that intra-vitreal bevacizumab may be a viable alternative treatment for the management of patients with DME and severe capillary loss. (Am J Ophthalmol 2009;147:1022-1030. (C) 2009 by Elsevier Inc. All rights reserved.)

Intraoperative bleeding during vitrectomy for diabetic tractional retinal detachment with versus without preoperative intravitreal bevacizumab (IBeTra study)

Aims: To compare the amount of intraoperative intraocular bleeding in patients with diabetes with macula-involving tractional retinal detachment (TRD) undergoing pars plana vitrectomy (PPV) with and without preoperative intravitreal bevacizumab (IVB) injection. Methods: An institutional study was carried out with consecutive patients with diabetic retinopathy and macula-involving TRD of recent (3 months) onset who were randomly assigned to PPV only (PPV group) or PPV combined with one IVB (1.5 mg/0.06 ml) injection 2 weeks prior to surgery (bevacizumab (BEV)/PPV group). All patients underwent 23-gauge PPV 3 weeks after baseline. The main outcome measure was erythrocyte count in the fluid retrieved from the vitrectomy cassette using a Neubauer counting chamber. Results: The study included 20 patients. The mean erythrocyte count was 14865x10(3) (SD 19332x10(3); median 4500x10(3)) cells in the BEV/PPV group, and 176240x10(3) (SD 108375x10(3); median 166600x10(3)) cells in the PPV group. The mean erythrocyte count was significantly lower in the BEV/PPV group than in the PPV group (p < 0.0001). No major adverse events were identified. Conclusion: Preoperative IVB injection was associated with reduced intraocular bleeding during 23-gauge PPV for diabetic macula-involving TRD. Further studies are needed to confirm our preliminary findings.

Intravitreal triamcinolone versus bevacizumab for treatment of refractory diabetic macular oedema (IBEME study)

Background/aims: The aim of this study was to compare the morphological and visual acuity outcomes associated with a single intravitreal injection of triamcinolone acetonide versus bevacizumab for the treatment of refractory diffuse diabetic macular oedema. Methods: Twenty-eight patients were randomly assigned to receive a single intravitreal injection of either 4 mg/0.1 ml triamcinolone acetonide or 1.5 mg/0.06 ml bevacizumab. Comprehensive ophthalmic evaluation was performed at baseline and at weeks 1, 4, 8 (+/- 1), 12 (+/- 2) and 24 (+/- 2) after treatment. Main outcome measures included central macular thickness measured with optical coherence tomography (OCT) and best corrected Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity. Results: Twenty-six patients (26 eyes) completed all study visits (two patients missed two consecutive study visits). Central macular thickness was significantly reduced in the intravitreal triamcinolone group compared with the bevacizumab group at weeks 4, 8, 12 and 24 (p<0.05). Logarithm of the minimum angle of resolution (LogMAR) best-corrected visual acuity was significantly higher at weeks 8 (0.69; similar to 20/100(+1)) and 12 (0.74; 20/100(-2)) in the intravitreal triamcinolone group compared with the bevacizumab group (weeks 8 (0.83; similar to 20/125(-1)) and 12 (0.86; 20/ 160(+2))) (p<0.05). Significant change from baseline in mean intraocular pressure (mmHg) was seen at week 4 (+2.25) only in the intravitreal triamcinolone group (p<0.0001). No patient had observed cataract progression during the study. Conclusions: One single intravitreal injection of triamcinolone may offer certain advantages over bevacizumab in the short-term management of refractory diabetic macular oedema, specifically with regard to changes in central macular thickness. The actual clinical relevance of our preliminary findings, however, remains to be determined in future larger studies.