Effect of intensive insulin therapy on macular biometrics, plasma VEGF and its soluble receptor in newly diagnosed diabetic patients.

BACKGROUND: To evaluate whether intensive insulin therapy leads to changes in macular biometrics (volume and thickness) in newly diagnosed diabetic patients with acute hyperglycaemia and its relationship with serum levels of vascular endothelial growth factor (VEGF) and its soluble receptor (sFlt-1). METHODS: Twenty-six newly diagnosed diabetic patients admitted to our hospital to initiate intensive insulin treatment were prospectively recruited. Examinations were performed on admission (day 1) and during follow-up (days 3, 10 and 21) and included a questionnaire regarding the presence of blurred vision, standardized refraction measurements and optical coherence tomography. Plasma VEGF and sFlt-1 were assessed by ELISA at baseline and during follow-up. RESULTS: At study entry seven patients (26.9%) complained of blurred vision and five (19.2%) developed burred vision during follow-up. Macular volume and thickness increased significantly (p = 0.008 and p = 0.04, respectively) in the group with blurred vision at day 3 and returned to the baseline value at 10 days. This pattern was present in 18 out of the 24 eyes from patients with blurred vision. By contrast, macular biometrics remained unchanged in the group without blurred vision. We did not detect any significant changes in VEGF levels during follow-up. By contrast, a significant reduction of sFlt-1 was observed in those patients with blurred vision at day 3 (p = 0.03) with normalization by day 10. CONCLUSION: Diabetic patients with blurred vision after starting insulin therapy present a significant transient increase in macular biometrics which is associated with a decrease in circulating sFlt-1.

The retinal pigment epithelium: something more than a constituent of the blood-retinal barrier--implications for the pathogenesis of diabetic retinopathy.

The retinal pigment epithelium (RPE) is an specialized epithelium lying in the interface between the neural retina and the choriocapillaris where it forms the outer blood-retinal barrier (BRB). The main functions of the RPE are the following: (1) transport of nutrients, ions, and water, (2) absorption of light and protection against photooxidation, (3) reisomerization of all-trans-retinal into 11-cis-retinal, which is crucial for the visual cycle, (4) phagocytosis of shed photoreceptor membranes, and (5) secretion of essential factors for the structural integrity of the retina. An overview of these functions will be given. Most of the research on the physiopathology of diabetic retinopathy has been focused on the impairment of the neuroretina and the breakdown of the inner BRB. By contrast, the effects of diabetes on the RPE and in particular on its secretory activity have received less attention. In this regard, new therapeutic strategies addressed to modulating RPE impairment are warranted.

Lipopolysaccharide-binding protein and soluble CD14 in the vitreous fluid of patients with proliferative diabetic retinopathy.

PURPOSE: The purpose of this study was to compare intravitreous levels of lipopolysaccharide-binding protein and soluble CD14 (sCD14) between patients with proliferative diabetic retinopathy (PDR) and nondiabetic subjects. METHODS: This study included 19 consecutive Type 2 diabetic patients with PDR in whom a vitrectomy was performed. Sixteen vitreous humors from nondiabetic patients matched by age, with idiopathic macular holes, were selected from our vitreous bank and used as a control group. Lipopolysaccharide-binding protein was assessed by enzyme-linked immunosorbent assay and sCD14 by a solid-phase enzyme-amplified sensitive immunoassay. RESULTS: Lipopolysaccharide-binding protein and sCD14 levels were significantly higher in patients with PDR than in the control group (lipopolysaccharide-binding protein, P < 0.001; sCD14, P < 0.01). After correcting for vitreal proteins, the results remained significantly higher in patients with PDR. No differences in serum levels were observed, and we did not find any correlation between serum and vitreous levels. A direct correlation between lipopolysaccharide-binding protein and sCD14 was detected in the vitreous fluid (r = 0.57; P < 0.001) but not in the plasma. Finally, a significant correlation between intravitreal levels of both lipopolysaccharide-binding protein and sCD14 and interleukin-8 and monocyte chemotactic protein-1 was also detected. CONCLUSION: Lipopolysaccharide-binding protein and sCD14 are elevated in the vitreous fluid of patients with PDR and thus may play a role in the innate immune response triggered by the inflammatory injury characteristic of PDR.

Effects of high glucose concentration on the barrier function and the expression of tight junction proteins in human retinal pigment epithelial cells.

There is no information on the direct effect of high glucose concentrations on the barrier function of retinal pigment epithelium (RPE). The aim of this study was to explore the effect of high glucose concentrations on the permeability and the expression of tight junction proteins (occludin, zonula occludens-1 (ZO-1) and claudin-1) in a human RPE line (ARPE-19). For this purpose, ARPE-19 cells were cultured for 3 weeks in a medium containing 5.5 mM D-glucose (mimicking physiological conditions) and 25 mM D-glucose (mimicking hyperglycemia that occurs in diabetic patients). The permeability was evaluated by measuring transepithelial electrical resistance (TER) and apical-basolateral movements of dextran. The expression of tight junction proteins was evaluated by real-time PCR (RT-PCR) and Western blot. Cells grown at 25 mM of D-glucose showed a significant higher TER and a significant lower dextran diffusion than the ones maintained at 5.5 mM of D-glucose. Occludin and ZO-1 mRNA levels and protein content were similar in cultures maintained in 5.5 mM and 25 mM D-glucose. By contrast, high glucose concentrations induced a significant overexpression of claudin-1 (mRNA: 1.03 +/- 0.48 vs 2.29 +/- 0.7 RQ; p = 0.039, at 21 days. Protein levels: 0.92 +/- 0.12 vs 1.14 +/- 0.28 arbitrary units; p = 0.03, at 21 days). However, after blocking claudin-1 expression using siRNA no changes in TER and permeability were observed. We conclude that high glucose concentration results in a reduction of permeability in ARPE-19 cells. In addition, our results suggest that the overexpression of claudin-1 induced by high glucose concentrations is not involved in the mechanisms by which glucose increases the tight junction sealing function. Further studies addressed to unravel the complexity of permeability regulation in RPE are needed.

High glucose concentration leads to differential expression of tight junction proteins in human retinal pigment epithelial cells.

INTRODUCTION: One of the early features of diabetic retinopathy is the breakdown of the blood-retinal barrier (BRB) due to disruption of the tight junctions. Whereas impairment of the proteins involved in the disruption of the tight junctions of the internal BRB has been extensively studied, there is no information on the direct effect of high glucose concentration on the barrier function of the outer blood-retinal barrier (formed by the retinal pigment epithelium [RPE]). The aim of this study was to explore the effect of high glucose concentration on the expression of tight junction proteins (occludin, zonula occludens-1 [ZO-1] and claudin-1) in a human RPE line under two distinct glucose concentrations. MATERIALS AND METHODS: An RPE cell line (ARPE-19) were cultured for 3 weeks in a medium supplemented with 10% fetal calf serum containing 5.5 mmol D-glucose (mimicking physiological conditions) or 25 mmol Dglucose (mimicking the hyperglycemia that occurs in diabetic patients). Occludin, ZO-1 and claudin-1 were studied by real-time polymerase chain reaction and Western blot at 14 and 21 days. RESULTS: Occludin and ZO-1 mRNA levels and protein content were similar in cultures maintained at 5.5 mmol and 25 mmol of D-glucose. In contrast, high glucose concentration (25 mmol) induced a clear upregulation in claudin-1 mRNA expression and protein content at 21 days (mRNA level: 1.03 vs 2.29; protein content: 0.92 vs 1.14). CONCLUSIONS: High glucose concentration leads to differential expression of tight junction proteins in ARPE-19 cells. In addition, our results suggest that the upregulation of claudin-1by glucose is involved in the increase of tight junction sealing function. The functional consequences and clinical applicability of these findings require further investigation.

Measuring permeability in human retinal epithelial cells (ARPE-19): implications for the study of diabetic retinopathy.

The retinal pigment epithelium (RPE) is a specialized epithelium lying in the interface between the -neural retina and the choriocapillaris where it forms the outer blood-retinal barrier (BRB). The tight junctions (TJ)s expressed in the outer BRB control fluids and solutes that enter the retina and this sealing function, which is essential for the retinal homeostasis, is impaired in diabetic retinopathy. In this -chapter, we provide the methods to explore the function of the RPE barrier by measuring Transepithelial electrical resistance (TER) and paracellular permeability to dextran in cultures of ARPE-19 cells (an immortalized RPE cell line). A method for inducing a lesion mimicking which occurs in diabetic retinopathy is described. In addition, methods for assessing mRNA expression and protein content of the main TJ proteins (occludin, zonula occludens-1 [ZO-1]) are detailed. Finally, we provide the methods required for confocal immunofluorescence detection of the TJ proteins, as well as for assessing the capacity of ARPE-19 cells to retain their functional properties.

Erythropoietin protects retinal pigment epithelial cells against the increase of permeability induced by diabetic conditions: essential role of JAK2/ PI3K signaling.

The outer blood-retinal barrier is formed by retinal pigment epithelial (RPE) cells and its disruption significantly contributes to the development of diabetic macular edema (DME). The aim of the study was to explore whether erythropoietin (Epo) has beneficial effects on the barrier function of human RPE cells and the main downstream pathways involved. ARPE-19 cells were cultured in standard conditions and under conditions leading to the disruption of the monolayer [25 mmol/L D-glucose plus IL-1ß (10 ng/mL)]. Epo (200 mU/mL/day) was added during the last 2 days of the experiment. The experiments were repeated in the presence of an Epo neutralizing antibody and specific inhibitors of JAK2 and PI3K (AG490 and LY294002, respectively). Permeability was evaluated by fluorescein isothiocyanate dextran (70 kDa) movements. Distribution of tight junction proteins was examined by immunofluorescence. Changes in cytosolic Ca(2+) induced by Epo were also measured. Epo treatment was able to prevent but not to restore the increase of permeability induced by high glucose plus IL-1ß. The protective effect of Epo on RPE barrier function was completely blocked by AG490 and almost completely abolished by LY294002. In addition, Epo was able to increase cytosolic Ca(2+) with dependence on extracellular calcium influx and this effect was blocked by either JAK2 or PI3K inhibition. We conclude that RPE disruption induced by high glucose plus IL-1ß is prevented by Epo through the downstream signaling of JAK2 and PI3K/AKT pathways.

Fenofibric acid reduces fibronectin and collagen type IV overexpression in human retinal pigment epithelial cells grown in conditions mimicking the diabetic milieu: functional implications in retinal permeability.

PURPOSE: To determine whether fenofibric acid (FA) reduces high glucose (HG)-induced basement membrane component overexpression and hyperpermeability in human retinal pigment epithelial (RPE) cells. METHODS: Retinal pigment epithelial cells (ARPE-19) were cultured for 18 days in normal glucose (5 mM) or HG (25 mM) medium and studied for the effects of FA on fibronectin (FN) and collagen IV (Coll IV) expression. During last 3 days of the experiment, 100 µM FA was added to cells grown in HG medium or in HG medium plus IL-1ß (HG + IL-1ß) to mimic, at least in part, the inflammatory aspect of the diabetic milieu. Real-time RT-PCR was performed to determine FN and Coll IV mRNA levels, whereas protein levels were assessed by Western blot analyses. Cell monolayer morphology and barrier function were analyzed by confocal microscopy using specific antibodies against tight junction proteins, ZO-1, and claudin-1 and by measuring apical-basolateral movements of FITC-dextran, respectively. RESULTS: FN and Coll IV expression were significantly increased in RPE cells grown in HG or HG + IL-1ß medium compared with cells grown in normal medium. When cells grown in HG or HG + IL-1ß medium were treated with FA, significant reductions in FN and Coll IV expression were observed. In addition, exposure to FA decreased excess permeability in a dose-dependent manner in cells grown in HG + IL-1ß medium. This effect was unrelated to changes in tight junction protein content. CONCLUSIONS: Findings from this study suggest that the downregulation of basement membrane components by FA may have a protective effect against outer blood-retinal barrier leakage associated with diabetic retinopathy.

Neurodegeneration: An early event of diabetic retinopathy.

Diabetic retinopathy (DR) has been classically considered to be a microcirculatory disease of the retina caused by the deleterious metabolic effects of hyperglycemia per se and the metabolic pathways triggered by hyperglycemia. However, retinal neurodegeneration is already present before any microcirculatory abnormalities can be detected in ophthalmoscopic examination. In other words, retinal neurodegeneration is an early event in the pathogenesis of DR which predates and participates in the microcirculatory abnormalities that occur in DR. Therefore, the study of the mechanisms that lead to neurodegeneration will be essential to identify new therapeutic targets in the early stages of DR. Elevated levels of glutamate and the overexpression of the renin- angiotensin-system play an essential role in the neurodegenerative process that occurs in diabetic retina. Among neuroprotective factors, pigment epithelial derived factor, somatostatin and erythropoietin seem to be the most relevant and these will be considered in this review. Nevertheless, it should be noted that the balance between neurotoxic and neuroprotective factors rather than levels of neurotoxic factors alone will determine the presence or absence of retinal neurodegeneration in the diabetic eye. New strategies, based on either the delivery of neuroprotective agents or the blockade of neurotoxic factors, are currently being tested in experimental models and in clinical pilot studies. Whether these novel therapies will eventually supplement or prevent the need for laser photocoagulation or vitrectomy awaits the results of additional clinical research.

Efecto de la hiperglucemia en la expresión de las proteínas de las tight junctions en células de epitelio pigmentario de la retina humana / High glucose concentration leads to differential expression of tight junction proteins in human retinal pigment epithelial cells

Introduction: One of the early features of diabetic retinopathy is the breakdown of the blood-retinal barrier (BRB) due to disruption of the tight junctions. Whereas impairment of the proteins involved in the disruption of the tight junctions of the internal BRB has been extensively studied, there is no information on the direct effect of high glucose concentration on the barrier function of the outer blood-retinal barrier (formed by the retinal pigment epithelium [RPE]). The aim of this study was to explore the effect of high glucose concentration on the expression of tight junction proteins (occludin, zonula occludens-1 [ZO-1] and claudin-1) in a human RPE line under two distinct glucose concentrations. Materials and methods: An RPE cell line (ARPE-19) were cultured for 3 weeks in a medium supplemented with 10% fetal calf serum containing 5.5 mmol D-glucose (mimicking physiological conditions) or 25 mmol D-glucose (mimicking the hyperglycemia that occurs in diabetic patients). Occludin, ZO-1 and claudin-1 were studied by real-time polymerase chain reaction and Western blot at 14 and 21 days. Results: Occludin and ZO-1 mRNA levels and protein content were similar in cultures maintained at 5.5 mmol and 25 mmol of D-glucose. In contrast, high glucose concentration (25 mmol) induced a clear upregulation in claudin-1 mRNA expression and protein content at 21 days (mRNA level: 1.03 vs 2.29; protein content: 0.92 vs 1.14). Conclusions: High glucose concentration leads to differential expression of tight junction proteins in ARPE-19 cells. In addition, our results suggest that the upregulation of claudin-1by glucose is involved in the increase of tight junction sealing function. The functional consequences and clinical applicability of these findings require further investigation (AU)

Introducción: Una de las primeras consecuencias de la retinopatía diabética es la rotura de la barrera hematorretiniana (BHR) causada por la interrupción de las tight junctions. Mientras que la alteración de las proteínas implicadas en la interrupción de las tight junctions de la BHR interna ha sido estudiada extensamente, la información sobre este proceso en la barrera hematorretiniana externa (constituida por el epitelio pigmentario de la retina) es escasa. El objetivo de este trabajo es estudiar el cambio de expresión (ARNm y proteína) de ocludina, zonula occludens-1 y claudina-1 en células de epitelio pigmentario de retina (EPR) humanas con dos concentraciones diferentes de glucosa. Materiales y métodos: Se utilizó una línea de células de EPR humano (ARPE-19), cultivada durante 3 semanas en un medio suplementado con un 10% de suero bovino fetal y con una concentración de 5,5 mmol de D-glucosa (simulando condiciones fisiológicas) o 25 mmol de D-glucosa (simulando la hiperglucemia que ocurre en pacientes diabéticos). Las proteínas de las tight junctions ocludina, zonula occludens-1 y claudina-1 se estudiaron por Western blot y PCR real time. Todas las determinaciones se hicieron a los 14 y 21 días. Resultados: La expresión de ARNm y proteína de ocludina y ZO-1 fue similar en cultivos mantenidos a 5,5 y 25 mmol de Dglucosa. Por el contrario, en condiciones de hiperglucemia (25 mmol) se produjo un claro aumento en la expresión de ARNm de la claudina-1 y en el contenido de esta proteína a los 21 días (concentraciones de ARNm, 1,03 frente a 2,29; proteína, 0,92 frente a 1,14) (..) (AU)