Glucose-induced increase in paracellular permeability and disruption of beta-receptor signaling in retinal endothelium.
Haselton F R,Dworska E J,Hoffman L H
Investigative ophthalmology & visual science
PURPOSE:To examine the effects of high glucose concentrations on retinal endothelial permeability in an in vitro model of the retinal microvasculature. METHODS:The permeability of the endothelial barrier to small solutes was measured in a chromatographic cell column consisting of bovine retinal endothelial cells cultured on porous fibronectin-coated microcarriers. In each cell column, permeability changes were evaluated by comparing the treatment permeability response over time with the initial baseline permeability. Short-term (2-hour) barrier effects of glucose were examined by measuring permeability at 15-minute intervals after an increase in perfusate concentration from baseline (5.5 mM) to high (25 mM) glucose. Long-term (to 57 days) effects were tested by addition of 25 mM glucose to microcarrier cultures. The effect of glucose on beta-receptor signaling was tested by measuring its effect on the permeability decrease produced by 1 microM isoproterenol. RESULTS:An increase from 5.5 mM to 25 mM glucose concentration did not change retinal endothelial cell monolayer permeability (n=6) during 2 hours. However, an increase in monolayer permeability was observed after 19 days (n=8) in the 25-mM glucose culture. Paralleling this time course, short-term exposure to 25 mM glucose did not prevent a decrease in permeability triggered by the beta-receptor agonist isoproterenol. However, the permeability effect of the agonist was blocked by long-term culture in 25 mM glucose. Permeability of retinal endothelial monolayers cultured in 5.5 mM glucose and treated with 1 microM isoproterenol decreased significantly to 0.71+/-0.06 of baseline (n=4; mean+/-SEM). However, permeability did not change in parallel cell columns made from microcarriers cultured in 25 mM glucose (0.97+/-0.2 of baseline permeability; n=4; mean+/-SEM). CONCLUSIONS:High-glucose culture decreases the retinal endothelial barrier and blocks the response to beta-adrenergic receptors. This model may prove valuable in exploring other hypotheses of increased permeability associated with diabetic retinopathy or other retinal diseases that break down the retinal vascular barrier.
Antioxidants attenuate early up regulation of retinal vascular endothelial growth factor in streptozotocin-diabetic rats.
Obrosova I G,Minchenko A G,Marinescu V,Fathallah L,Kennedy A,Stockert C M,Frank R N,Stevens M J
Diabetologia
AIMS/HYPOTHESIS:A strong positive correlation has been found between lipid peroxidation product and vascular endothelial growth factor concentrations in the vitreous of patients with proliferative diabetic retinopathy. To establish a causal relation between diabetes-associated enhanced oxidative stress and vascular endothelial growth factor production, we evaluated two antioxidants, DL-alpha-lipoic acid and taurine, on retinal vascular endothelial growth factor protein and mRNA expression and on parameters of oxidative stress in streptozotocin-diabetic rats. METHODS:Our experiments were on control rats and streptozotocin-diabetic rats with a 6-week duration of diabetes, treated with or without DL-alpha-lipoic acid (100 mg x kg(-1) x d(-1), i.p.) or taurine (1% in the diet) starting from induction of diabetes. Vascular endothelial growth factor protein in retinal homogenates was assessed by sandwich ELISA with an affinity-purified polyclonal antibody and vascular endothelial growth factor mRNA by ribonuclease protection assay. Retinal lipid peroxidation products i.e. malondialdehyde plus 4-hydroxyalkenals were quantified with N-methyl-2-phenylindole. Retinal reduced and oxidized glutathione, ascorbate, dehydroascorbate, and sorbitol pathway intermediates were measured spectrofluorometrically, and taurine by reverse-phase HPLC. RESULTS:Vascular endothelial growth factor protein concentration (means +/- SD) was increased in diabetic rats compared with control rats (33+/-7 vs 19+/-5 pg/mg total protein, p < 0.01) This increase was attenuated by taurine (26+/-8, p < 0.05) and prevented by DL-alpha-lipoic acid (21+/-4, p < 0.01). Vascular endothelial growth factor mRNA abundance was reduced by 1.4-fold in diabetic rats compared with control rats and this decrease was attenuated but not completely prevented by both antioxidants. Malondialdehyde plus 4-hydroxyalkenal concentration was increased in diabetic rats compared with control rats, and both antioxidants arrested accumulation of lipid peroxidation products. Taurine, reduced glutathione, oxidized glutathione, ascorbate, dehydroascorbate and sorbitol pathway intermediate concentrations as well as oxidized glutathione/reduced glutathione and dehydroascorbate/ascorbate ratios were similar in control and diabetic rats treated with or without taurine. CONCLUSION/INTERPRETATION:Oxidative stress is directly involved in up regulation of vascular endothelial growth factor protein in the retina during early diabetes.
10.1007/s001250100631
Human pericyte-endothelial cell interactions in co-culture models mimicking the diabetic retinal microvascular environment.
Tarallo Sonia,Beltramo Elena,Berrone Elena,Porta Massimo
Acta diabetologica
Pericytes regulate vascular tone, perfusion pressure and endothelial cell (EC) proliferation in capillaries. Thiamine and benfotiamine counteract high glucose-induced damage in vascular cells. We standardized two human retinal pericyte (HRP)/EC co-culture models to mimic the diabetic retinal microvascular environment. We aimed at evaluating the interactions between co-cultured HRP and EC in terms of proliferation/apoptosis and the possible protective role of thiamine and benfotiamine against high glucose-induced damage. EC and HRP were co-cultured in physiological glucose and stable or intermittent high glucose, with or without thiamine/benfotiamine. No-contact model: EC were plated on a porous membrane suspended into the medium and HRP on the bottom of the same well. Cell-to-cell contact model: EC and HRP were plated on the opposite sides of the same membrane. Proliferation (cell counts and DNA synthesis), apoptosis and tubule formation in Matrigel were assessed. In the no-contact model, stable high glucose reduced proliferation of co-cultured EC/HRP and EC alone and increased co-cultured EC/HRP apoptosis. In the contact model, both stable and intermittent high glucose reduced co-cultured EC/HRP proliferation and increased apoptosis. Stable high glucose had no effects on HRP in separate cultures. Both EC and HRP proliferated better when co-cultured. Thiamine and benfotiamine reversed high glucose-induced damage in all cases. HRP are sensitive to soluble factors released by EC when cultured in high glucose conditions, as suggested by conditioned media assays. In the Matrigel models, addition of thiamine and benfotiamine re-established the high glucose-damaged interactions between EC/HRP and stabilized microtubules.
10.1007/s00592-012-0390-5
Diabetes-induced changes in retinal NAD-redox status: pharmacological modulation and implications for pathogenesis of diabetic retinopathy.
Obrosova I G,Stevens M J,Lang H J
Pharmacology
Diabetes-induced changes in retinal metabolism and function have been linked to increased aldose reductase activity, hypoxia or 'pseudohypoxia' (increase in NADH/NAD+ attributed to increased sorbitol dehydrogenase activity). To address this controversy, we evaluated the effects of two vasoactive compounds, alpha(1)-adrenoceptor antagonist prazosin and antioxidant DL-alpha-lipoic acid, as well as sorbitol dehydrogenase inhibitor (SDI-157) and aldose reductase inhibitor (sorbinil) on retinal free mitochondrial and cytosolic NAD+/NADH ratios in streptozotocin-diabetic rats. Diabetes-induced decrease in mitochondrial and cytosolic NAD+/NADH ratios was completely or partially corrected by prazosin and DL-alpha-lipoic acid (despite the fact that prazosin did not affect and DL-alpha-lipoic acid even further increased sorbitol pathway activity) as well as by sorbinil, whereas SDI-157 was totally ineffective. Hypoxia-like metabolic changes in the diabetic retina originate from aldose reductase, but not sorbitol dehydrogenase activity.
10.1159/000056091
Resveratrol improves diabetic retinopathy possibly through oxidative stress - nuclear factor κB - apoptosis pathway.
Soufi Farhad Ghadiri,Mohammad-Nejad Daryoush,Ahmadieh Hamid
Pharmacological reports : PR
BACKGROUND:This study was designed to investigate the possible effectiveness of resveratrol (trans-3,5,4'-trihydroxystilbene) administration on oxidative stress, nuclear factor κB (NF-κB) activity and apoptosis rate in streptozotocin-nicotinamide-induced diabetic retinopathy. METHODS:Male Wistar rats were divided into four groups: normal control, diabetic control, normal rats treated with resveratrol, and diabetic rats treated with resveratrol. Diabetes was induced by injection of streptozotocin (50 mg/kg; ip), 15 min after the prescription of nicotinamide (110 mg/kg; ip) in 12 h fasted rats. RESULTS:Four-month oral resveratrol administration (5 mg/kg/day) significantly alleviated hyperglycemia, weight loss, enhancement of oxidative markers (lipid peroxidation index and oxidized to reduced glutathione ratio) and superoxide dismutase activity in both blood and retinas of the diabetic rats. Moreover, resveratrol administration to diabetic rats improved the elevated levels of retinas NF-κB activity and apoptosis rate. On the other hand, four months resveratrol administration prevented from disarrangement and reduction in thickness of retinal layers. CONCLUSION:These beneficial antidiabetic observations suggest that resveratrol may be considered as a therapeutic supplement to prevent from diabetic retinopathy.
10.1016/s1734-1140(12)70948-9
Effects of an Aquaporin 4 Inhibitor, TGN-020, on Murine Diabetic Retina.
Oosuka Shou,Kida Teruyo,Oku Hidehiro,Horie Taeko,Morishita Seita,Fukumoto Masanori,Sato Takaki,Ikeda Tsunehiko
International journal of molecular sciences
PURPOSE:To investigate the effect of a selective aquaporin 4 (AQP4) inhibitor, 2-(nicotinamide)-1,3,4-thiadiazole (TGN-020), on the expression of vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) production, as well as on the retinal edema in diabetic retina. METHODS:Intravitreal injections of bevacizumab, TGN-020, or phosphate-buffered saline (PBS) were performed on streptozotocin-induced diabetic rats. Retinal sections were immunostained for anti-glial fibrillary acidic protein (GFAP), anti-AQP4, and anti-VEGF. Protein levels of VEGF from collected retinas were determined by Western blot analysis. In addition, retinal vascular leakage of Evans Blue was observed in the flat-mounted retina from the diabetic rats in the presence or absence of TGN-020. Volumetric changes of rat retinal Müller cells (TR-MUL5; transgenic rat Müller cells) and intracellular levels of ROS were determined using flow cytometry analysis of ethidium fluorescence in the presence or absence of TGN-020 or bevacizumab under physiological and high glucose conditions. RESULTS:In the diabetic retina, the immunoreactivity and protein levels of VEGF were suppressed by TGN-020. AQP4 immunoreactivity was higher than in the control retinas and the expressions of AQP4 were co-localized with GFAP. Similarly to VEGF, AQP4 and GFAP were also suppressed by TGN-020. In the Evans Blue assay, TGN-020 decreased leakage in the diabetic retinas. In the cultured Müller cells, the increase in cell volumes and intracellular ROS production under high glucose condition were suppressed by exposure to TGN-020 as much as by exposure to bevacizumab. CONCLUSION:TGN-020 may have an inhibitory effect on diabetic retinal edema.
10.3390/ijms21072324
Neuroprotective Effects of Nicotinamide (Vitamin B) on Neurodegeneration in Diabetic Rat Retinas.
Jung Kyoung In,Han Jeong-Sun,Park Chan Kee
Nutrients
The loss of inner retinal neurons is an initial event in diabetic retinopathy. In diabetic retinas, oxidative stress is increased, which could lead to increased oxidative DNA damage. Nicotinamide is a precursor to nicotinamide adenine dinucleotide, which contributes to the DNA damage response. We investigated whether nicotinamide plays a neuroprotective role in diabetic retinal neurodegeneration in terms of DNA repair. Male Sprague Dawley rats with streptozotocin-induced diabetes were orally administered nicotinamide (500 mg/kg/day) for 4 or 12 weeks. Oxidative stress exhibited by dihydroethidium was upregulated at 4 and 12 weeks after onset of diabetes, and nicotinamide treatment reduced oxidative stress at 4 weeks after induction of diabetes. Oxidative DNA damage measured by 8-hydroxy-2'-deoxyguanosine (8-OHdG) increased at 4 and 12 weeks after induction of diabetes and decreased following nicotinamide treatment. The elevated expression of glial fibrillary acidic protein (GFAP) induced by diabetes was attenuated by nicotinamide treatment. In Western blot analysis, the increased expression of cleaved PARP-1 in diabetes was attenuated by nicotinamide treatment at 12 weeks after induction of diabetes. The diabetes-induced apoptosis of inner retinal cells detected by the TUNEL assay was reduced by nicotinamide treatment. In conclusion, nicotinamide attenuated retinal neurodegeneration in diabetes, probably by reducing oxidative DNA damage and supporting DNA repair.
10.3390/nu14061162