Placental growth factor-1 and epithelial haemato-retinal barrier breakdown: potential implication in the pathogenesis of diabetic retinopathy.

AIMS/HYPOTHESIS: Disruption of the retinal pigment epithelial (RPE) barrier contributes to sub-retinal fluid and retinal oedema as observed in diabetic retinopathy. High placental growth factor (PLGF) vitreous levels have been found in diabetic patients. This work aimed to elucidate the influence of PLGF-1 on a human RPE cell line (ARPE-19) barrier in vitro and on normal rat eyes in vivo. METHODS: ARPE-19 permeability was measured using transepithelial resistance and inulin flux under stimulation of PLGF-1, vascular endothelial growth factor (VEGF)-E and VEGF 165. Using RT-PCR, we evaluated the effect of hypoxic conditions or insulin on transepithelial resistance and on PLGF-1 and VEGF receptors. The involvement of mitogen-activated protein kinase (MEK, also known as MAPK)/extracellular signal-regulated kinase (ERK, also known as EPHB2) signalling pathways under PLGF-1 stimulation was evaluated by western blot analysis and specific inhibitors. The effect of PLGF-1 on the external haemato-retinal barrier was evaluated after intravitreous injection of PLGF-1 in the rat eye; evaluation was by semi-thin analysis and zonula occludens-1 immunolocalisation on flat-mounted RPE. RESULTS: In vitro, PLGF-1 induced a reversible decrease of transepithelial resistance and enhanced tritiated inulin flux. These effects were specifically abolished by an antisense oligonucleotide directed at VEGF receptor 1. Exposure of ARPE-19 cells to hypoxic conditions or to insulin induced an upregulation of PLGF-1 expression along with increased transcellular permeability. The PLGF-1-induced RPE cell permeability involved the MEK signalling pathway. Injection of PLGF-1 in the rat eye vitreous induced an opening of the RPE tight junctions with subsequent sub-retinal fluid accumulation, retinal oedema and cytoplasm translocation of junction proteins. CONCLUSIONS/INTERPRETATION: Our results indicate that PLGF-1 may be a potential regulation target for the control of diabetic retinal and macular oedema.

Integrating quantitative proteomics and metabolomics in a cellular model of diabetic retinopathy

Diabetic retinopathy is the leading cause of visual loss in individuals under the age of 55. Most investigations into the pathogenesis of diabetic retinopathy have been concentrated on the neural retina since this is where clinical lesions are manifested. Recently, however, various abnormalities in the structural and secretory functions of retinal pigment epithelium that are essential for neuroretina survival, have been found in diabetic retinopathy. In this context, here we study the effect of hyperglycemic and hypoxic conditions on the metabolism of a human retinal pigment epithelial cell line (ARPE-19) by integrating quantitative proteomics using tandem mass tagging (TMT), untargeted metabolomics using MS and NMR, and 13C-glucose isotopic labeling for metabolic tracking. We observed a remarkable metabolic diversification under our simulated in vitro hyperglycemic conditions of diabetes, characterized increased flux through polyol pathways and inhibition of the Krebs cycle and oxidative phosphorylation. Importantly, under low oxygen supply RPE cells seem to consume rapidly glycogen storages and stimulate anaerobic glycolysis. Our results therefore pave the way to future scenarios involving new therapeutic strategies addressed to modulating RPE metabolic impairment, with the aim of regulating structural and secretory alterations of RPE. Finally, this study shows the importance of tackling biomedical problems by integrating metabolomic and proteomics results.

Eye Fundus Image Analysis for Automatic Detection of Diabetic Retinopathy

Diabetes is a rapidly increasing worldwide problem which is characterised by defective metabolism of glucose that causes long-term dysfunction and failure of various organs. The most common complication of diabetes is diabetic retinopathy (DR), which is one of the primary causes of blindness and visual impairment in adults. The rapid increase of diabetes pushes the limits of the current DR screening capabilities for which the digital imaging of the eye fundus (retinal imaging), and automatic or semi-automatic image analysis algorithms provide a potential solution. In this work, the use of colour in the detection of diabetic retinopathy is statistically studied using a supervised algorithm based on one-class classification and Gaussian mixture model estimation. The presented algorithm distinguishes a certain diabetic lesion type from all other possible objects in eye fundus images by only estimating the probability density function of that certain lesion type. For the training and ground truth estimation, the algorithm combines manual annotations of several experts for which the best practices were experimentally selected. By assessing the algorithm’s performance while conducting experiments with the colour space selection, both illuminance and colour correction, and background class information, the use of colour in the detection of diabetic retinopathy was quantitatively evaluated. Another contribution of this work is the benchmarking framework for eye fundus image analysis algorithms needed for the development of the automatic DR detection algorithms. The benchmarking framework provides guidelines on how to construct a benchmarking database that comprises true patient images, ground truth, and an evaluation protocol. The evaluation is based on the standard receiver operating characteristics analysis and it follows the medical practice in the decision making providing protocols for image- and pixel-based evaluations. During the work, two public medical image databases with ground truth were published: DIARETDB0 and DIARETDB1. The framework, DR databases and the final algorithm, are made public in the web to set the baseline results for automatic detection of diabetic retinopathy. Although deviating from the general context of the thesis, a simple and effective optic disc localisation method is presented. The optic disc localisation is discussed, since normal eye fundus structures are fundamental in the characterisation of DR.

Diabetic retinopathy is associated with early autonomic dysfunction assessed by exercise-related heart rate changes

Diabetic retinopathy has been associated with cardiac autonomic dysfunction in both type 1 and type 2 diabetes mellitus (DM) patients. Heart rate (HR) changes during exercise testing indicate early alterations in autonomous tonus. The aim of the present study was to investigate the association of diabetic retinopathy with exercise-related HR changes. A cross-sectional study was performed on 72 type 2 and 40 type 1 DM patients. Autonomic dysfunction was assessed by exercise-related HR changes (Bruce protocol). The maximum HR increase, defined as the difference between the peak exercise rate and the resting rate at baseline, and HR recovery, defined as the reduction in HR from the peak exercise to the HR at 1, 2, and 4 min after the cessation of the exercise, were determined. In type 2 DM patients, lower maximum HR increase (OR = 1.62, 95%CI = 1.03-2.54; P = 0.036), lower HR recovery at 2 (OR = 2.04, 95%CI = 1.16-3.57; P = 0.012) and 4 min (OR = 2.67, 95%CI = 1.37-5.20; P = 0.004) were associated with diabetic retinopathy, adjusted for confounding factors. In type 1 DM, the absence of an increase in HR at intervals of 10 bpm each during exercise added 100% to the odds for diabetic retinopathy (OR = 2.01, 95%CI = 1.1-3.69; P = 0.02) when adjusted for DM duration, A1c test and diastolic blood pressure. In conclusion, early autonomic dysfunction was associated with diabetic retinopathy. The recognition of HR changes during exercise can be used to identify a high-risk group for diabetic retinopathy.

Neuron-Derived Semaphorin 3A is an Early Inducer of Vascular Permeability in Diabetic Retinopathy

La détérioration de la barrière hémato rétinienne et l'oedème maculaire consécutif est une manifestation cardinale de la rétinopathie diabétique (RD) et la caractéristique clinique la plus étroitement associée à la perte de la vue. Alors que l'oedème maculaire affecte plus de 25% des patients souffrant de diabète, les modalités de traitement actuellement disponibles tels que les corticostéroïdes administrés localement et les thérapies anti-VEGF récemment approuvés présentent plusieurs inconvénients. Bien que le lien entre une rupture de l’unité neuro-vasculaire et la pathogénèse de la RD ait récemment été établi, l’influence de la signalisation neuro-vasculaire sur la vasculopathie oculaire diabetique a jusqu’à présent reçu peu d’attention. Ici, à l’aide d’ètudes humaines et animales, nous fournissons la première preuve du rôle essentiel de la molécule de guidage neuronale classique Sémaphorine 3A dans l’instigation de la perméabilité vasculaire maculaire pathologique dans le diabète de type 1. L’étude de la dynamique d’expression de Sémaphorine 3A révèle que cette dernière est induite dans les phases précoces hyperglycèmiques du diabète dans la rétine neuronale et participe à la rupture initiale de la fonction de barrière endothéliale. En utilisant le modèle de souris streptozotocine pour simuler la rétinopathie diabétique humaine, nous avons démontré par une série d’approches analogue que la neutralisation de Sémaphorine 3A empêche de façon efficace une fuite vasculaire rétinienne. Nos résultats identifient une nouvelle cible thérapeutique pour l’oedème maculaire diabétique en plus de fournir d’autres preuves de communication neuro-vasculaire dans la pathogènese de la RD.

MULLER CELLS AND DIABETIC-RETINOPATHY

Muller cells provide nutrition for neural cells. We studied the structure and ultrastructure of Muller cells in the retina of thirty 3-month old Wistar rats; divided equally into 3 groups: normal rats, alloxan diabetic rats and treated alloxan diabetic rats. 1 and 12 months after induction of diabetes. We observed that the Muller cell nuclei under light microscope examination had hexagonal shape and higher density than the other nuclei. Differences between groups could be observed only by electron microscopy. In the diabetic rats, Muller cells presented dispersion of nuclear chromatin and electrondense nuclear granulations, with the presence of increased glycogen, dense bodies and lysosomes in the cytoplasm. The alterations were more frequent in the perivascular region and at 12 months. The treated diabetic rats exhibited some alterations we observed in diabetic rats. but these alterations were less intense. We conclude that, despite the treatment, the diabetic retinopathy continues to evolve.

Müller cells and diabetic retinopathy.

Müller cells provide nutrition for neural cells. We studied the structure and ultrastructure of Müller cells in the retina of thirty 3-month old Wistar rats, divided equally into 3 groups: normal rats, alloxan diabetic rats and treated alloxan diabetic rats, 1 and 12 months after induction of diabetes. We observed that the Müller cell nuclei under light microscope examination had hexagonal shape and higher density than the other nuclei. Differences between groups could be observed only by electron microscopy. In the diabetic rats, Müller cells presented dispersion of nuclear chromatin and electrondense nuclear granulations, with the presence of increased glycogen, dense bodies and lysosomes in the cytoplasm. The alterations were more frequent in the perivascular region and at 12 months. The treated diabetic rats exhibited some alterations we observed in diabetic rats, but these alterations were less intense. We conclude that, despite the treatment, the diabetic retinopathy continues to evolve.

Prevalence of Diabetes and Diabetic Retinopathy in a Brazilian Population

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Retinal overexpression of angiopoietin-2 mimics diabetic retinopathy and enhances vascular damages in hyperglycemia

Our previous data suggested that angiopoietin-2 (Ang-2) is linked to pericyte loss, thereby playing an important role in diabetic retinopathy. In this study, we investigated the effect of retinal overexpression of human Ang-2 (mOpsinhAng2 mouse) on vascular morphology in non-diabetic and streptozotozin-induced diabetic animals. Pericyte (PC) coverage and acellular capillary (AC) formation were quantitated in retinal digest preparations after 3 and 6 months of diabetes duration. The degree of retinopathy in non-diabetic mOpsinhAng2 mice at 3 months (-21% PC, +49% AC) was comparable to age-matched diabetic wild type mice. Diabetic mOpsinhAng2 mice exhibited significantly worse vascular pathology than wild type counterparts at 6 months. Quantitative PCR revealed that human Ang-2 mRNA was highly overexpressed in retinas of transgenic mice. Our data demonstrate that overexpression of Ang-2 in the retina enhances vascular pathology, indicating that Ang-2 plays an essential role in diabetic vasoregression via destabilization of pericytes.

Endothelin antagonism prevents diabetic retinopathy in NOD mice: a potential role of the angiogenic factor adrenomedullin

Altered activity of retinal endothelin-1 (ET-1) and nitric oxide may play a causal role in the hemodynamic and histopathological changes of diabetic retinopathy. This study evaluated the therapeutic potential of long-term selective blockade of the ET-1(A) receptor (ETRA) to prevent the development of retinopathy in a genetic mouse model of nonobese type 1 diabetes (NOD). Mice with NOD that received subcutaneous implantation of insulin pellets and wild-type control mice were treated for 4 months with the selective ETRA antagonist LU208075 (30 mg/kg/day) via drinking water. At the end of the study, blood glucose levels were evaluated, and animals were anesthetized and perfused intracardially with FITC-labeled dextran. Retinas were removed and either fixed in formalin for confocal microscope evaluation of retinal vascular filling or transferred to RNALater for quantitative reverse transcriptase-polymerase chain reaction to evaluate expression of NOS-3, NOS-1, ET-1, ETRA, ETRB, and the angiogenic factor adrenomedullin. Compared with wild-type controls, expression of ET-1, ETRA, ETRB, and adrenomedullin in mice with NOD were markedly upregulated in the retinas of nontreated mice (cycle time values relative to GAPDH [deltaCt], 14.8 vs. 13.7, 18.57 vs. 17.5, 10.76 vs. 9.9, and 11.7 vs. 9.1, respectively). Mean integral fluorescence intensity (MIFI) of retinal vascular filling was reduced from normal values of 24 to 12.5 in nontreated animals. LU208075 treatment normalized the upregulated expression of ET-1 and adrenomedullin, as well as the deficit in MIFI, but did not affect the increased ETRA and ETRB expression or the elevated plasma glucose levels found in nontreated animals. NOS isoform expression was essentially unchanged. ETRA antagonists may provide a novel therapeutic strategy to slow or prevent progression of retinal microvascular damage and proliferation in patients for whom there is clear evidence of activation of the ET-1 system.

Early loss of arteriolar smooth muscle cells: more than just a pericyte loss in diabetic retinopathy

Incipient diabetic retinopathy is characterized by increased capillary permeability and progressive capillary occlusion. The earliest structural change is the loss of pericytes (PC) from the retinal capillaries. With the availability of the XLacZ mouse, which expresses the LacZ reporter in a PC/vascular smooth muscle cell (vSMC) specific fashion, we quantitatively assessed the temporal dynamics of smooth muscle cells in arterioles under hyperglycemic conditions. We induced stable hyperglycemia in XLacZ mice. After 4, 8, and 12 weeks of diabetes retinae were isolated and beta-galactosidase/lectin stained. The numbers of smooth muscle cells were counted in retinal whole mounts, and diameters of retinal radial and branching arterioles and venules were analyzed at different distances apart from the center of the retina. After eight weeks of diabetes, the numbers of vSMCs were significantly reduced in radial arterioles 1000 microm distant from the optic disc. At proximal sites of branching arterioles (400 microm distant from the center), and at distal sites (1000 microm), vSMC were significantly reduced already after 4 weeks (to a maximum of 31 %). These changes were not associated with any measurable variation in vessel diameters. These data indicate quantitatively that hyperglycemia not only causes pericyte loss, but also loss of vSMCs in the retinal vasculature. Our data suggest that arteriolar vSMC in the eye underlie similar regulations which induce early pericyte loss in the diabetic retina.

Angiopoietin-2 causes pericyte dropout in the normal retina: evidence for involvement in diabetic retinopathy

Pericyte loss is an early pathologic feature of diabetic retinopathy, consistently present in retinae of diabetic humans and animals. Because pericyte recruitment and endothelial cell survival are controlled, in part, by the angiopoietin/Tie2 ligand/receptor system, we studied the expression of angiopoietin-2 and -1 in relation to the evolution of pericyte loss in diabetic rat retinae, using quantitative retinal morphometry, and in retinae from mice with heterozygous angiopoietin deficiency (Ang-2 LacZ knock-in mice). Finally, recombinant angiopoietin-2 was injected into eyes of nondiabetic rats, and pericyte numbers were quantitated in retinal capillaries. Angiopoietin-1 protein was present in the normal maturing retina and was upregulated 2.5-fold in diabetic retinae over 3 months of diabetes. In contrast, angiopoietin-2 protein was consistently upregulated more than 30-fold in the retinae of diabetic rats, preceding the onset of pericyte loss. Heterozygous angiopoietin-2 deficiency completely prevented diabetes-induced pericyte loss and reduced the number of acellular capillary segments. Injection of angiopoietin-2 into the eyes of normal rats induced a dose-dependent pericyte loss. These data show that upregulation of angiopoietin-2 plays a critical role in the loss of pericytes in the diabetic retina.

Pericyte migration: a novel mechanism of pericyte loss in experimental diabetic retinopathy

OBJECTIVE: The mechanism underlying pericyte loss during incipient diabetic retinopathy remains controversial. Hyperglycemia induces angiopoietin-2 (Ang-2) transcription, which modulates capillary pericyte coverage. In this study, we assessed loss of pericyte subgroups and the contribution of Ang-2 to pericyte migration. RESEARCH DESIGN AND METHODS: Numbers of total pericytes and their subgroups were quantified in retinal digest preparations of spontaneous diabetic XLacZ mice. Pericytes were divided into subgroups according to their localization, their position relative to adjacent endothelial cells, and the expression of LacZ. The contribution of Ang-2 to pericyte migration was assessed in Ang-2 overexpressing (mOpsinhAng2) and deficient (Ang2LacZ) mice. RESULTS: Pericyte numbers were reduced by 16% (P < 0.01) in XLacZ mice after 6 months of diabetes. Reduction of pericytes was restricted to pericytes on straight capillaries (relative reduction 27%, P < 0.05) and was predominantly observed in LacZ-positive pericytes (-20%, P < 0.01). Hyperglycemia increased the numbers of migrating pericytes (69%; P < 0.05), of which the relative increase due to diabetes was exclusively in LacZ-negative pericytes, indicating reduced adherence to the capillaries (176%; P < 0.01). Overexpression of Ang-2 in nondiabetic retinas mimicked diabetic pericyte migration of wild-type animals (78%; P < 0.01). Ang-2 deficient mice completely lacked hyperglycemia-induced increase in pericyte migration compared with wild-type littermates. CONCLUSIONS: Diabetic pericyte loss is the result of pericyte migration, and this process is modulated by the Ang-Tie system.

Activity of the glycosylating enzyme, core 2 GlcNAc (beta1,6) transferase, is higher in polymorphonuclear leukocytes from diabetic patients compared with age-matched control subjects: relevance to capillary occlusion in diabetic retinopathy

The exact mechanism for capillary occlusion in diabetic retinopathy is still unclear, but increased leukocyte-endothelial cell adhesion has been implicated. We examined the possibility that posttranslational modification of surface O-glycans by increased activity of core 2 transferase (UDP-Glc:Galbeta1-3GalNAcalphaRbeta-N-acetylglucoaminyltr ansferase) is responsible for increased adhesion of leukocytes to vascular endothelium in diabetes. The mean activity of core 2 transferase in polymorphonuclear leukocytes isolated from type 1 and type 2 diabetic patients was higher compared with age-matched control subjects (1,638 +/- 91 [n = 42] vs. 249 +/- 35 pmol x h(-1) x mg(-1) protein [n = 24], P = 0.00013; 1,459 +/- 194 [n = 58] vs. 334 +/- 86 [n = 11], P = 0.01). As a group, diabetic patients with retinopathy had significantly higher mean activity of core 2 transferase compared with individuals with no retinopathy. There was a significant association between enzyme activity and severity of retinopathy in type 1 and type 2 diabetic patients. There was a strong correlation between activity of core 2 transferase and extent of leukocyte adhesion to cultured retinal capillary endothelial cells for diabetic patients but not for age-matched control subjects. Results from transfection experiments using human myelocytic cell line (U937) demonstrated a direct relationship between increased activity of core 2 transferase and increased binding to cultured endothelial cells. There was no relationship between activity of core 2 transferase and HbA(1c) (P = 0.8314), serum advanced glycation end product levels (P = 0.4159), age of the patient (P = 0.7896), and duration of diabetes (P = 0.3307). On the basis that branched O-glycans formed by the action of core 2 transferase participate in leukocyte adhesion, the present data suggest the involvement of this enzyme in increased leukocyte-endothelial cell adhesion and the pathogenesis of capillary occlusion in diabetic retinopathy.