Advances in our understanding of diabetic retinopathy

Diabetic retinopathy remains the most common complication of diabetes mellitus and is a leading cause of visual loss in industrialized nations. The clinicopathology of the diabetic retina has been extensively studied, although the precise pathogenesis and cellular and molecular defects that lead to retinal vascular, neural and glial cell dysfunction remain somewhat elusive. This lack of understanding has seriously limited the therapeutic options available for the ophthalmologist and there is a need to identify the definitive pathways that initiate retinal cell damage and drive progression to overt retinopathy. The present review begins by outlining the natural history of diabetic retinopathy, the clinical features and risk factors. Reviewing the histopathological data from clinical specimens and animal models, the recent paradigm that neuroretinal dysfunction may play an important role in the early development of the disease is discussed. The review then focuses on the molecular pathogenesis of diabetic retinopathy with perspective provided on new advances that have furthered our understanding of the key mechanisms underlying early changes in the diabetic retina. Studies have also emerged in the past year suggesting that defective repair of injured retinal vessels by endothelial progenitor cells may contribute to the pathogenesis of diabetic retinopathy. We assess these findings and discuss how they could eventually lead to new therapeutic options for diabetic retinopathy.

Diabetic-like loss of corneal sensitivity in the 50% galactose fed rat is ameliorated with topical administration of an aldose reductase inhibitor

Purpose: To investigate the temporal course of corneal sensitivity loss & the role of aldose reductase inhibitors (ARI) in an animal model of diabetic ocular complications. Methods: Weanling male S-D rats were randomly grouped to received ad libitum water & diet consisting of Purina (#5001) w/ either: 50% starch (CON,n=15) or 50% D-galactose (GAL,n=30). Half the galactosemic rats (ARI,n=15) received topical 0.25% CT-112 (3x daily, 20µl, Senju Pharmaceutical Co., Japan). Control & remaining half of the galactosemic animals received equivalent doses of saline eyedrops. Rats were restrained w/o medication during sensitivity measurements conducted w/ a Cochet-Bonnet Aesthesiometer mounted on a micromanipulator. The end of the filament (0.012mm dia.), which applied a mean pressure of 0.96 g/mm perpendicular to the corneal surface at center, was in the plane of focus of a slit-lamp biomicroscope. Measurements were conducted by two investigators which were masked to the treatment group. The average blink-responses from 10 consecutive stimuli to each cornea were expressed as a percent. Results: Mean (±SD) baseline corneal sensitivity in all groups were similar (CON 73%±11, GAL 71%±15, ARI 74%±16). Corneal sensitivity in the galactosemic rat was decreased (p

Fetal and maternal leptin in pre-gestational diabetic pregnancy

Objective: To compare maternal and fetal leptin among women without diabetes, women with type 1 diabetes, and women with type 2 diabetes. 

Methods: In a prospective study at the National Maternity Hospital, Dublin, 40 women with type 1 diabetes, 10 with type 2 diabetes, and 30 without diabetes were enrolled between July 2006 and July 2008. Maternal (36-week) and cord blood leptin was measured by enzyme-linked immunoassay. 

Results: No difference was found in maternal leptin among the groups: without diabetes (mean, range): 325 pg/mL, 36-1492 pg/mL; type 1 diabetes: 343.2 pg/mL, 55.5-1108.2 pg/mL; type 2 diabetes: 2022 pg/mL, 35.1-1553.3 pg/mL (P>0.05). Leptin levels were higher among fetuses of women with type 1 (223 pg/mL, 25.7-810 pg/mL) and type 2 (447.2 pg/mL, 1363-679 pg/mL) diabetes than among women without diabetes (803 pg/mL, 273-623.1 pg/mL; P<0.05). The single significant predictor of fetal leptin for the whole cohort was maternal body mass index (BMI; r=039, P=0.01). Only third-trimester glycosylated hemoglobin (HbA1c) was significantly related to fetal leptin after controlling for maternal BMI among women with diabetes (r=028, P=0.04).

 Conclusion: Fetuses of women with diabetes might have some degree of leptin resistance. This might be important in appetite regulation in extrauterine life. (C) 2012 International Federation of Gynecology and Obstetrics. 

Activation of the ACE2/Angiotensin-(1-7)/Mas Receptor Axis Enhances the Reparative Function of Dysfunctional Diabetic Endothelial Progenitors

We tested the hypothesis that activation of the protective arm of the renin angiotensin system, the angiotensin-converting enzyme 2 (ACE2)/angiotensin-(1-7) [Ang-(1-7)]/Mas receptor axis, corrects the vasoreparative dysfunction typically seen in the CD34(+) cells isolated from diabetic individuals. Peripheral blood CD34(+) cells from patients with diabetes were compared with those of nondiabetic controls. Ang-(1-7) restored impaired migration and nitric oxide bioavailability/cGMP in response to stromal cell-derived factor and resulted in a decrease in NADPH oxidase activity. The survival and proliferation of CD34(+) cells from diabetic individuals were enhanced by Ang-(1-7) in a Mas/phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner. ACE2 expression was lower, and ACE2 activators xanthenone and diminazine aceturate were less effective in inducing the migration in cells from patients with diabetes compared with controls. Ang-(1-7) overexpression by lentiviral gene modification restored both the in vitro vasoreparative functions of diabetic cells and the in vivo homing efficiency to areas of ischemia. A cohort of patients who remained free of microvascular complications despite having a history of longstanding inadequate glycemic control had higher expression of ACE2/Mas mRNA than patients with diabetes with microvascular complications matched for age, sex, and glycemic control. Thus, ACE2/Ang-(1-7)\Mas pathway activation corrects existing diabetes-induced CD34(+) cell dysfunction and also confers protection from development of this dysfunction.

Current treatments in diabetic macular oedema:systematic review and meta-analysis

The aim of this systematic review is to appraise the evidence for the use of anti-VEGF drugs and steroids in diabetic macular oedema (DMO) as assessed by change in best corrected visual acuity (BCVA), central macular thickness and adverse events

Anti-angiogenic factors and pre-eclampsia in type 1 diabetic women

Aims/hypothesis: Elevated anti-angiogenic factors such as soluble fms-like tyrosine kinase 1 (sFlt1), a soluble form of vascular endothelial growth factor receptor, and endoglin, a co-receptor for TGFß1, confer high risk of pre-eclampsia in healthy pregnant women. In this multicentre prospective study, we determined levels of these and related factors in pregnant women with type 1 diabetes, a condition associated with a fourfold increase in pre-eclampsia.
Methods: Maternal serum sFlt1, endoglin, placental growth factor (PlGF) and pigment epithelial derived factor were measured in 151 type 1 diabetic and 24 healthy non-diabetic women at each trimester and at term.
Results: Approximately 22% of the diabetic women developed pre-eclampsia, primarily after their third trimester visit. In women with pre-eclampsia (diabetic pre-eclampsia, n?=?26) vs those without hypertensive complications (diabetic normotensive, n?=?95), significant changes in angiogenic factors were observed, predominantly in the early third trimester and prior to clinical manifestation of pre-eclampsia. Serum sFlt1 levels were increased approximately twofold in type 1 diabetic pre-eclampsia vs type 1 diabetic normotensive women at the third trimester visit (p?<?0.05) and the normal rise of PlGF during pregnancy was blunted (p?<?0.05). Among type 1 diabetic women, third trimester sFlt1 and PlGF were inversely related (r2?=?42%, p?<?0.0001). Endoglin levels were increased significantly in the diabetic group as a whole vs the non-diabetic group (p?<?0.0001).
Conclusions/interpretation: Higher sFlt1 levels, a blunted PlGF rise and an elevated sFlt1/PlGF ratio are predictive of pre-eclampsia in pregnant women with type 1 diabetes. Elevated endoglin levels in women with type 1 diabetes may confer a predisposition to pre-eclampsia and may contribute to the high incidence of pre-eclampsia in this patient group.

Haplotype association analysis of genes within the WNT signalling pathways in diabetic nephropathy

Background: Renal interstitial fibrosis and glomerular sclerosis are hallmarks of diabetic nephropathy (DN) and several studies have implicated members of the WNT pathways in these pathological processes. This study comprehensively examined common genetic variation within the WNT pathway for association with DN.

Methods: Genes within the WNT pathways were selected on the basis of nominal significance and consistent direction of effect in the GENIE meta-analysis dataset. Common SNPs and common haplotypes were examined within the selected WNT pathway genes in a white population with type 1 diabetes, discordant for DN (cases: n = 718; controls: n = 749). SNPs were genotyped using Sequenom or Taqman assays. Association analyses were performed using PLINK, to compare allele and haplotype frequencies in cases and controls. Correction for multiple testing was performed by either permutation testing or using false discovery rate.

Results: A logistic regression model including collection centre, duration of diabetes, and average HbA1c as covariates highlighted three SNPs in GSK3B (rs17810235, rs17471, rs334543), two in DAAM1 (rs1253192, rs1252906) and one in NFAT5 (rs17297207) as being significantly (P< 0.05) associated with DN, however these SNPs did not remain significant after correction for multiple testing. Logistic regression of haplotypes, with ESRD as the outcome, and pairwise interaction analyses did not yield any significant results after correction for multiple testing.

Conclusions: These results indicate that both common SNPs and common haplotypes of WNT pathway genes are not strongly associated with DN. However, this does not completely exclude these or the WNT pathways from association with DN, as unidentified rare genetic or copy number variants could still contribute towards the genetic architecture of DN.© 2013 Kavanagh et al.; licensee BioMed Central Ltd.

Cardiomyopathy and Diastolic Dysfunction in the Embryo and Neonate of a Type 1 Diabetic Mouse Model

Objective: The purpose of this study was to examine the effect of maternal type 1 diabetes on the structure and function of the embryonic and neonatal mouse heart.

Methods: Type 1 diabetes was induced in female C57BL6/J mice using streptozotocin. Embryonic (n = 105) and neonatal hearts (n = 46) were examined using high-frequency ultrasound (US) and a cohort of E18.5 (n = 34) and 1-day-old pup hearts (n = 27) underwent histological examination.

Results: Global cardiac hypertrophy in late gestation (E18.5) was evident on US in the diabetic group compared to controls with increased interventricular septal (IVS) thickness (0.44 ± 0.08 mm vs 0.36 ± 0.08 mm, P < .05) and increased left ventricular wall thickness (0.38 ± 0.04 mm vs 0.29 mm ± 0.05, P < .01). Isovolumetric relaxation time was initially prolonged in the diabetic group but resolved by E18.5 to control values. Histological examination at E18.5 demonstrated increased transverse measurements (2.42 ± 0.72 mm/g vs 1.86 ± 0.55 mm/g, P < .05) and increased IVS thickness (0.64 ± 0.20 mm/g vs 0.43 ± 0.15 mm/g, P < .05) in diabetic embryos compared to control embryos.

Conclusion: Maternal hyperglycemia has severe effects on offspring with evidence of cardiac impairment and cardiac hypertrophy in the embryo. These effects persisted in the 1-day old but attenuated in the 1-week old suggesting cardiac remodeling after the hyperglycemic milieu of pregnancy is removed

Insulin receptor substrate-2 is expressed in kidney epithelium and up-regulated in diabetic nephropathy

Diabetic nephropathy (DN) is a progressive fibrotic condition that may lead to end-stage renal disease and kidney failure. Transforming growth factor-ß1 and bone morphogenetic protein-7 (BMP7) have been shown to induce DN-like changes in the kidney and protect the kidney from such changes, respectively. Recent data identified insulin action at the level of the nephron as a crucial factor in the development and progression of DN. Insulin requires a family of insulin receptor substrate (IRS) proteins for its physiological effects, and many reports have highlighted the role of insulin and IRS proteins in kidney physiology and disease. Here, we observed IRS2 expression predominantly in the developing and adult kidney epithelium in mouse and human. BMP7 treatment of human kidney proximal tubule epithelial cells (HK-2 cells) increases IRS2 transcription. In addition, BMP7 treatment of HK-2 cells induces an electrophoretic shift in IRS2 migration on SDS/PAGE, and increased association with phosphatidylinositol-3-kinase, probably due to increased tyrosine/serine phosphorylation. In a cohort of DN patients with a range of chronic kidney disease severity, IRS2 mRNA levels were elevated approximately ninefold, with the majority of IRS2 staining evident in the kidney tubules in DN patients. These data show that IRS2 is expressed in the kidney epithelium and may play a role in the downstream protective events triggered by BMP7 in the kidney. The specific up-regulation of IRS2 in the kidney tubules of DN patients also indicates a novel role for IRS2 as a marker and/or mediator of human DN progression.

Effects of modified LDL and HDL on retinal pigment epithelial cells: a role in diabetic retinopathy?

Aims/hypothesis: Blood–retina barrier leakage in diabetes results in extravasation of plasma lipoproteins. Intra-retinal modified LDLs have been implicated in diabetic retinopathy (DR), but their effects on retinal pigment epithelial (RPE) cells and the added effects of extravasated modified HDLs 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 heavilyoxidised 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.

Therapeutic effects of PPARα agonists on diabetic retinopathy in type 1 diabetes models

Retinal vascular leakage, inflammation, and neovascularization (NV) are features of diabetic retinopathy (DR). Fenofibrate, a peroxisome proliferator-activated receptor a (PPARa) agonist, has shown robust protective effects against DR in type 2 diabetic patients, but its effects on DR in type 1 diabetes have not been reported. This study evaluated the efficacy of fenofibrate on DR in type 1 diabetes models and determined if the effect is PPARa dependent. Oral administration of fenofibrate significantly ameliorated retinal vascular leakage and leukostasis in streptozotocin-induced diabetic rats and in Akita mice. Favorable effects on DR were also achieved by intravitreal injection of fenofibrate or another specific PPARa agonist. Fenofibrate also ameliorated retinal NV in the oxygen-induced retinopathy (OIR) model and inhibited tube formation and migration in cultured endothelial cells. Fenofibrate also attenuated overexpression of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and vascular endothelial growth factor (VEGF) and blocked activation of hypoxia-inducible factor-1 and nuclear factor-?B in the retinas of OIR and diabetic models. Fenofibrate's beneficial effects were blocked by a specific PPARa antagonist. Furthermore, Ppara knockout abolished the fenofibrate-induced downregulation of VEGF and reduction of retinal vascular leakage in DR models. These results demonstrate therapeutic effects of fenofibrate on DR in type 1 diabetes and support the existence of the drug target in ocular tissues and via a PPARa-dependent mechanism.

Mechanisms of modified LDL-induced pericyte loss and retinal injury in diabetic retinopathy

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 LDLinduced 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.