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1.
Diabetologia ; 64(3): 693-706, 2021 03.
Article in English | MEDLINE | ID: mdl-33319325

ABSTRACT

AIMS/HYPOTHESIS: Diabetic retinopathy is characterised by retinal neurodegeneration and retinal vascular abnormalities, affecting one third of diabetic patients with disease duration of more than 10 years. Accumulated evidence suggests that serine racemase (SR) and D-serine are correlated with the pathogenesis of diabetic retinopathy and the deletion of the Srr gene reverses neurovascular pathologies in diabetic mice. Since D-serine content is balanced by SR synthesis and D-amino acid oxidase (DAAO) degradation, we examined the roles of DAAO in diabetic retinopathy and further explored relevant therapy. METHODS: Rats were used as a model of diabetes by i.p. injection of streptozotocin at the age of 2 months and blood glucose was monitored with a glucometer. Quantitative real-time PCR was used to examine Dao mRNA and western blotting to examine targeted proteins in the retinas. Bisulphite sequencing was used to examine the methylation of Dao mRNA promoter in the retinas. Intravitreal injection of DAAO-expressing adenovirus (AAV8-DAAO) was conducted one week before streptozotocin administration. Brain specific homeobox/POU domain protein 3a (Brn3a) immunofluorescence was conducted to indicate retinal ganglion cells at 3 months after virus injection. The permeability of the blood-retinal barrier was examined by Evans blue leakage from retinal capillaries. Periodic acid-Schiff staining and haematoxylin counterstaining were used to indicate retinal vasculature, which was further examined with double immunostaining at 7 months after virus injection. RESULTS: At the age of 12 months, DAAO mRNA and protein levels in retinas from diabetic animals were reduced to 66.2% and 70.4% of those from normal (control) animals, respectively. The Dao proximal promoter contained higher levels of methylation in diabetic than in normal retinas. Consistent with the observation, DNA methyltransferase 1 was increased in diabetic retinas. Injection of DAAO-expressing virus completely prevented the loss of retinal ganglion cells and the disruption of blood-retinal barrier in diabetic rats. Diabetic retinas contained retinal ganglion cells at a density of 54 ± 4/mm2, which was restored to 68 ± 9/mm2 by DAAO overexpression, similar to the levels in normal retinas. The ratio between the number of endothelial cells and pericytes in diabetic retinas was 6.06 ± 1.93/mm2, which was reduced to 3.42 ± 0.55/mm2 by DAAO overexpression; the number of acellular capillaries in diabetic retinas was 10 ± 5/mm2, which was restored to 6 ± 2/mm2 by DAAO overexpression, similar to the levels in normal retinas. Injection of the DAAO-expressing virus increased the expression of occludin and reduced gliosis, which were examined to probe the mechanism by which the disrupted blood-retinal barrier in diabetic rats was rescued and retinal neurodegeneration was prevented. CONCLUSIONS/INTERPRETATION: Altogether, overexpression of DAAO before the onset of diabetes protects against neurovascular abnormalities in retinas from diabetic rats, which suggests a novel strategy for preventing diabetic retinopathy. Graphical abstract.


Subject(s)
Blood-Retinal Barrier/enzymology , D-Amino-Acid Oxidase/biosynthesis , Diabetic Retinopathy/prevention & control , Retinal Ganglion Cells/enzymology , Animals , Blood-Retinal Barrier/pathology , Capillary Permeability , D-Amino-Acid Oxidase/genetics , DNA Methylation , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/enzymology , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/enzymology , Diabetic Retinopathy/enzymology , Diabetic Retinopathy/etiology , Diabetic Retinopathy/pathology , Enzyme Induction , Male , Nerve Degeneration , Promoter Regions, Genetic , Rats, Sprague-Dawley , Retinal Ganglion Cells/pathology , Transcription Factor Brn-3A/genetics , Transcription Factor Brn-3A/metabolism
2.
Int J Mol Sci ; 21(5)2020 Feb 27.
Article in English | MEDLINE | ID: mdl-32121029

ABSTRACT

Blood-retinal barrier (BRB) dysfunction represents one of the most significant changes occurring during diabetic retinopathy. We set up a high-reproducible human-based in vitro BRB model using retinal pericytes, retinal astrocytes, and retinal endothelial cells in order to replicate the human in vivo environment with the same numerical ratio and layer order. Our findings showed that high glucose exposure elicited BRB breakdown, enhanced permeability, and reduced the levels of junction proteins such as ZO-1 and VE-cadherin. Furthermore, an increased expression of pro-inflammatory mediators (IL-1ß, IL-6) and oxidative stress-related enzymes (iNOS, Nox2) along with an increased production of reactive oxygen species were observed in our triple co-culture paradigm. Finally, we found an activation of immune response-regulating signaling pathways (Nrf2 and HO-1). In conclusion, the present model mimics the closest human in vivo milieu, providing a valuable tool to study the impact of high glucose in the retina and to develop novel molecules with potential effect on diabetic retinopathy.


Subject(s)
Astrocytes/metabolism , Blood-Retinal Barrier/metabolism , Endothelial Cells/metabolism , Glucose/metabolism , Pericytes/metabolism , Retina/metabolism , Antigens, CD/metabolism , Biomarkers/metabolism , Blood-Retinal Barrier/enzymology , Cadherins/metabolism , Coculture Techniques , Glucose/pharmacology , Heme Oxygenase-1/metabolism , Humans , In Vitro Techniques , Inflammation/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Models, Biological , NADPH Oxidase 2/genetics , NADPH Oxidase 2/metabolism , NF-E2-Related Factor 2/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Oxidative Stress/genetics , Protein Serine-Threonine Kinases/metabolism , Reactive Oxygen Species/metabolism , Zonula Occludens-1 Protein/metabolism , NF-kappaB-Inducing Kinase
3.
FASEB J ; 32(5): 2539-2548, 2018 05.
Article in English | MEDLINE | ID: mdl-29263022

ABSTRACT

Inflammation plays an important role in the pathogenesis of diabetic retinopathy. We have previously demonstrated the effect of cathepsin D (CD) on the mechanical disruption of retinal endothelial cell junctions and increased vasopermeability, as well as increased levels of CD in retinas of diabetic mice. Here, we have also examined the effect of CD on endothelial-pericyte interactions, as well as the effect of dipeptidyl peptidase-4 (DPP4) inhibitor on CD in endothelial-pericyte interactions in vitro and in vivo. Cocultured cells that were treated with pro-CD demonstrated a significant decrease in the expression of platelet-derived growth factor receptor-ß, a tyrosine kinase receptor that is required for pericyte cell survival; N-cadherin, the key adherens junction protein between endothelium and pericytes; and increases in the vessel destabilizing agent, angiopoietin-2. The effect was reversed in cells that were treated with DPP4 inhibitor along with pro-CD. With pro-CD treatment, there was a significant increase in the phosphorylation of the downstream signaling protein, PKC-α, and Ca2+/calmodulin-dependent protein kinase II in endothelial cells and pericytes, which disrupts adherens junction structure and function, and this was significantly reduced with DPP4 inhibitor treatment. Increased CD levels, vasopermeability, and alteration in junctional-related proteins were observed in the retinas of diabetic rats, which were significantly changed with DPP4 inhibitor treatment. Thus, DPP4 inhibitors may be used as potential adjuvant therapeutic agents to treat increased vascular leakage observed in patients with diabetic macular edema.-Monickaraj, F., McGuire, P., Das, A. Cathepsin D plays a role in endothelial-pericyte interactions during alteration of the blood-retinal barrier in diabetic retinopathy.


Subject(s)
Blood-Retinal Barrier/enzymology , Cathepsin D/metabolism , Cell Communication , Diabetic Retinopathy/enzymology , Endothelial Cells/enzymology , Pericytes/enzymology , Angiopoietin-2/metabolism , Animals , Blood-Retinal Barrier/pathology , Cadherins/metabolism , Calcium Signaling/drug effects , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Capillary Permeability/drug effects , Cathepsin D/antagonists & inhibitors , Cell Survival/drug effects , Cyclic AMP-Dependent Protein Kinases/metabolism , Diabetic Retinopathy/pathology , Endothelial Cells/pathology , Male , Nerve Tissue Proteins/metabolism , Pericytes/pathology , Rats , Rats, Sprague-Dawley , Receptor, Platelet-Derived Growth Factor beta/metabolism
4.
Biochem Pharmacol ; 142: 145-154, 2017 10 15.
Article in English | MEDLINE | ID: mdl-28651842

ABSTRACT

Diabetic retinopathy is characterized by the breakdown of endothelial blood-retinal barrier. We tested the hypothesis that sulodexide (SDX), a highly purified glycosaminoglycan composed of 80% iduronylglycosaminoglycan sulfate and 20% dermatan sulfate, protects human retinal endothelial cells (HREC) from high glucose (HG)-induced damage, through the suppression of inflammatory ERK/cPLA2/COX-2/PGE2 pathway, by blocking the effect of advanced glycation end-products (AGEs). HREC were treated with HG (25mM) or AGEs (glycated-BSA, 2mg/ml) for 48h, with or without SDX (60µg/ml) or aflibercept (AFL, 40µg/ml), a VEGF-trap. SDX protected HREC from HG-induced damage (MTT and LDH release) and preserved their blood-retinal barrier-like properties (Trans Endothelial Electrical Resistance and junction proteins, claudin-5, VE-cadherin and occludin, immunofluorescence and immunoblot) as well as their angiogenic potential (Tube Formation Assay). Both HG and AGEs increased phosphoERK and phospho-cPLA2, an effect counteracted by SDX and, less efficiently, by AFL. Both HG and exogenous VEGF (80ng/ml) increased PGE2 release, an effect partially reverted by SDX for HG and by AFL for VEGF. Analysis of NFκB activity revealed that HG increased the abundance of p65 in the nuclear fraction (nuclear translocation), an effect entirely reverted by SDX, but only partially by AFL. SDX, AFL and SDX+AFL protected HREC even when added 24h after HG. These data show that SDX protects HREC from HG damage and suggest that it counteracts the activation of ERK/cPLA2/COX-2/PGE2 pathway by reducing AGE-related signaling and downstream NFκB activity. This mechanism, partially distinct from VEGF blockade, may contribute to the therapeutic effect of SDX.


Subject(s)
Blood-Retinal Barrier/drug effects , Cyclooxygenase 2/metabolism , Glycation End Products, Advanced/metabolism , Glycosaminoglycans/pharmacology , Phospholipases A2/metabolism , Receptor for Advanced Glycation End Products/metabolism , Vascular Endothelial Growth Factor A/metabolism , Blood-Retinal Barrier/enzymology , Blood-Retinal Barrier/metabolism , Cell Survival/drug effects , Cells, Cultured , Diabetic Retinopathy/metabolism , Diabetic Retinopathy/prevention & control , Glycosaminoglycans/isolation & purification , Humans , Primary Cell Culture
5.
Diab Vasc Dis Res ; 14(3): 200-213, 2017 05.
Article in English | MEDLINE | ID: mdl-28301218

ABSTRACT

Using a porcine model of diabetes mellitus and hypercholesterolaemia, we previously showed that diabetes mellitus and hypercholesterolaemia is associated with a chronic increase in blood-brain barrier permeability in the cerebral cortex, leading to selective binding of immunoglobulin G and deposition of amyloid-beta1-42 peptide in pyramidal neurons. Treatment with Darapladib (GlaxoSmithKline, SB480848), an inhibitor of lipoprotein-associated phospholipase-A2, alleviated these effects. Here, investigation of the effects of chronic diabetes mellitus and hypercholesterolaemia on the pig retina revealed a corresponding increased permeability of the blood-retina barrier coupled with a leak of plasma components into the retina, alterations in retinal architecture, selective IgG binding to neurons in the ganglion cell layer, thinning of retinal layers due to cell loss and increased glial fibrillary acidic protein expression in Müller cells, all of which were curtailed by treatment with Darapladib. These findings suggest that chronic diabetes mellitus and hypercholesterolaemia induces increased blood-retina barrier permeability that may be linked to altered expression of blood-retina barrier-associated tight junction proteins, claudin and occludin, leading to structural changes in the retina consistent with diabetic retinopathy. Additionally, results suggest that drugs with vascular anti-inflammatory properties, such as Darapladib, may have beneficial effects on eye diseases strongly linked to vascular abnormalities such as diabetic retinopathy and age-related macular degeneration.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/antagonists & inhibitors , Anti-Inflammatory Agents/pharmacology , Benzaldehydes/pharmacology , Blood-Retinal Barrier/drug effects , Capillary Permeability/drug effects , Diabetes Mellitus, Experimental/drug therapy , Diabetic Retinopathy/prevention & control , Hypercholesterolemia/drug therapy , Oximes/pharmacology , Phospholipase A2 Inhibitors/pharmacology , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Animals , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/enzymology , Blood-Brain Barrier/pathology , Blood-Brain Barrier/physiopathology , Blood-Retinal Barrier/enzymology , Blood-Retinal Barrier/pathology , Blood-Retinal Barrier/physiopathology , Claudin-5/metabolism , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/enzymology , Diabetes Mellitus, Experimental/physiopathology , Diabetic Retinopathy/enzymology , Diabetic Retinopathy/etiology , Diabetic Retinopathy/physiopathology , Gliosis , Hypercholesterolemia/complications , Hypercholesterolemia/enzymology , Hypercholesterolemia/physiopathology , Immunoglobulin G/metabolism , Male , Occludin/metabolism , Protein Binding , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/enzymology , Sus scrofa
6.
Sci Rep ; 5: 12796, 2015 Aug 05.
Article in English | MEDLINE | ID: mdl-26242473

ABSTRACT

Neural vascular barrier is essential for the life of multicellular organisms, and its impairment by tissue hypoxia is known to be a central of pathophysiology accelerating the progression of various intractable neural diseases. Therefore, the molecules involved in hypoxia-induced impairment of vascular barrier can be the targets to establish new therapies for intractable diseases. Here, we demonstrate that a disintegrin and metalloproteinases (ADAMs) 12 and 17 expressed in endothelial cells are the molecules responsible for the impairment of neural vascular barrier by hypoxia. Brain microvascular endothelial cells in vitro lost their barrier properties immediately after hypoxic stimulation through diminished localization of claudin-5, a tight junction molecule, on cell membranes. Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17. Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the in vivo neural vasculature under hypoxia from the loss of barrier function. This is the first report to specify the molecules which are responsible for hypoxia-induced impairment of neural vascular barrier and furthermore can be the targets of new therapeutic strategies for intractable neural diseases.


Subject(s)
ADAM Proteins/physiology , Endothelial Cells/enzymology , ADAM12 Protein , ADAM17 Protein , Animals , Blood-Retinal Barrier/cytology , Blood-Retinal Barrier/enzymology , Cell Hypoxia , Cell Line , Cell Membrane/metabolism , Claudin-5/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/enzymology , Male , Mice , Mice, Inbred C57BL , Retina/enzymology , Retinal Vessels/cytology , Retinal Vessels/enzymology
7.
Am J Pathol ; 184(2): 541-55, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24326256

ABSTRACT

Blood-retinal barrier (BRB) breakdown and related vascular changes are implicated in several ocular diseases. The molecules and mechanisms regulating BRB integrity and pathophysiology are not fully elucidated. Caveolin-1 (Cav-1) ablation results in loss of caveolae and microvascular pathologies, but the role of Cav-1 in the retina is largely unknown. We examined BRB integrity and vasculature in Cav-1 knockout mice and found a significant increase in BRB permeability, compared with wild-type controls, with branch veins being frequent sites of breakdown. Vascular hyperpermeability occurred without apparent alteration in junctional proteins. Such hyperpermeability was not rescued by inhibiting eNOS activity. Veins of Cav-1 knockout retinas exhibited additional pathological features, including i) eNOS-independent enlargement, ii) altered expression of mural cell markers (eg, down-regulation of NG2 and up-regulation of αSMA), and iii) dramatic alterations in mural cell phenotype near the optic nerve head. We observed a significant NO-dependent increase in retinal artery diameter in Cav-1 knockout mice, suggesting that Cav-1 plays a role in autoregulation of resistance vessels in the retina. These findings implicate Cav-1 in maintaining BRB integrity in retinal vasculature and suggest a previously undefined role in the retinal venous system and associated mural cells. Our results are relevant to clinically significant retinal disorders with vascular pathologies, including diabetic retinopathy, uveoretinitis, and primary open-angle glaucoma.


Subject(s)
Blood-Retinal Barrier/metabolism , Blood-Retinal Barrier/pathology , Caveolin 1/deficiency , Retinal Vein/metabolism , Retinal Vein/pathology , Animals , Biomarkers/metabolism , Blood-Retinal Barrier/enzymology , Blood-Retinal Barrier/ultrastructure , Caveolin 1/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide Synthase Type III/metabolism , Permeability , Phenotype , Protein Transport , Retinal Vein/enzymology , Retinal Vein/ultrastructure , Tight Junction Proteins/metabolism
8.
Biochem Pharmacol ; 86(11): 1603-13, 2013 Dec 01.
Article in English | MEDLINE | ID: mdl-24076420

ABSTRACT

Diabetic retinopathy is one of the leading causes of blindness and the most common complication of diabetes with no cure available. We investigated the role of phospholipases A2 (PLA2) in diabetic retinopathy using an in vitro blood-retinal barrier model (BRB) and an in vivo streptozotocin (STZ)-induced diabetic model. Mono- and co-cultures of endothelial cells (EC) and pericytes (PC), treated with high or fluctuating concentrations of glucose, to mimic the diabetic condition, were used. PLA2 activity, VEGF and PGE2 levels and cell proliferation were measured, with or without PLA2 inhibition. Diabetes was induced in rats by STZ injection and PLA2 activity along with VEGF, TNFα and ICAM-1 levels were measured in retina. High or fluctuating glucose induced BRB breakdown, and increased PLA2 activity, PGE2 and VEGF in EC/PC co-cultures; inhibition of PLA2 in mono- or co-cultures treated with high or fluctuating glucose dampened PGE2 and VEGF production down to the levels of controls. High or fluctuating glucose increased EC number and reduced PC number in co-cultures; these effects were reversed after transfecting EC with small interfering RNA targeted to PLA2. PLA2 and COX-2 protein expressions were significantly increased in microvessels from retina of diabetic rats. Diabetic rats had also high retinal levels of VEGF, ICAM-1 and TNFα that were reduced by treatment with a cPLA2 inhibitor. In conclusion, the present findings indicate that PLA2 upregulation represents an early step in glucose-induced alteration of BRB, possibly upstream of VEGF; thus, PLA2 may be an interesting target in managing diabetic retinopathy.


Subject(s)
Blood-Retinal Barrier/enzymology , Diabetes Mellitus, Experimental/enzymology , Diabetic Retinopathy/enzymology , Phospholipases A2/metabolism , Animals , Blood-Retinal Barrier/immunology , Blood-Retinal Barrier/pathology , Cattle , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Coculture Techniques , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/immunology , Diabetes Mellitus, Experimental/pathology , Diabetic Retinopathy/etiology , Diabetic Retinopathy/immunology , Diabetic Retinopathy/pathology , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Glucose/pharmacology , Male , Microscopy, Confocal , Pericytes/cytology , Pericytes/drug effects , Pericytes/enzymology , Phospholipase A2 Inhibitors/pharmacology , Phospholipases A2/biosynthesis , Rats , Rats, Sprague-Dawley , Streptozocin/pharmacology , Vascular Endothelial Growth Factor A/metabolism
9.
Microvasc Res ; 82(3): 346-50, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21945644

ABSTRACT

OBJECTIVE: To determine the efficacy of pazopanib eye drops in the streptozotocin induced diabetic retinopathy rat model. METHODS: A 0.5% w/v pazopanib suspension was prepared in phosphate buffered saline (PBS, pH 7.4) in the presence of 0.5% w/v sodium carboxymethyl cellulose. Brown Norway rats were divided into three groups (n=4) - (1) healthy, (2) diabetic, and (3) diabetic with treatment. The drug suspension was administered twice daily as eye drops to group 3 for 30 days. Efficacy parameters including the number of adherent leukocytes in the retinal vasculature (leukostasis), blood-retinal FITC-dextran leakage, and vitreous-to-plasma protein ratio were measured. RESULTS: Pazopanib suspension in the form of eye drops significantly reduced leukostasis (32%), FITC-dextran leakage (39%), and the vitreous-to-plasma protein ratio (64%) in diabetic animals compared to untreated diabetic group. CONCLUSION: Pazopanib eye drops can alleviate retinal complications of diabetic retinopathy.


Subject(s)
Blood-Retinal Barrier/drug effects , Capillary Permeability/drug effects , Diabetes Mellitus, Experimental/drug therapy , Diabetic Retinopathy/prevention & control , Leukostasis/prevention & control , Macular Edema/prevention & control , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Sulfonamides/pharmacology , Administration, Ophthalmic , Animals , Blood Glucose/metabolism , Blood Proteins/metabolism , Blood-Retinal Barrier/enzymology , Body Weight , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/enzymology , Diabetic Retinopathy/enzymology , Diabetic Retinopathy/etiology , Indazoles , Leukocytes/drug effects , Leukocytes/enzymology , Leukostasis/enzymology , Leukostasis/etiology , Macular Edema/enzymology , Macular Edema/etiology , Male , Molecular Targeted Therapy , Ophthalmic Solutions , Protein Kinase Inhibitors/administration & dosage , Protein-Tyrosine Kinases/metabolism , Pyrimidines/administration & dosage , Rats , Rats, Inbred BN , Sulfonamides/administration & dosage , Time Factors , Vitreous Body/metabolism
10.
Am J Pathol ; 176(3): 1517-24, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20110406

ABSTRACT

Vision loss in diabetic retinopathy is due to macular edema characterized by increased vascular permeability, which involves phosphorylation associated with activation of protein kinase C (PKC) isoforms. Herein, we demonstrated PKC delta inhibition could prevent blood-retinal barrier breakdown in diabetic retinopathy. Increased vascular permeability of diabetic retina was accompanied by a decrease of zonula occludens (ZO)-1 and ZO-2 expression. In diabetic retina and advanced glycation end product-treated human retinal microvascular endothelial cells, vascular leakage and loss of ZO-1 and ZO-2 on retinal vessels were effectively restored or prevented with treatment of rottlerin, transfection of PKC-delta-DN, or siRNA for PKC delta. Interestingly, PKC delta translocated from cytosol to membrane in advanced glycation end product-treated human retinal microvascular endothelial cells, which was blocked by PKC delta inhibition. Taken together, PKC delta activation, related to its subcellular translocation, is involved in vascular permeability in response to diabetes, and inhibition of PKC delta effectively restores loss of tight junction proteins in retinal vessels. Therefore, we suggest that inhibition of PKC delta could be an alternative treatment to blood-retinal barrier breakdown in diabetic retinopathy.


Subject(s)
Blood-Retinal Barrier/enzymology , Blood-Retinal Barrier/pathology , Diabetic Retinopathy/enzymology , Diabetic Retinopathy/pathology , Protein Kinase C-delta/antagonists & inhibitors , Animals , Blood-Retinal Barrier/drug effects , Blood-Retinal Barrier/physiopathology , Capillary Permeability/drug effects , Cytosol/drug effects , Cytosol/enzymology , Diabetic Retinopathy/physiopathology , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Endothelial Cells/pathology , Genes, Dominant/genetics , Humans , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Microvessels/pathology , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Protein Kinase C-delta/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Transport/drug effects , RNA, Small Interfering/metabolism , Retinal Vessels/pathology , Tight Junctions/drug effects , Tight Junctions/metabolism , Vascular Endothelial Growth Factor A/pharmacology
11.
Lab Invest ; 85(5): 597-607, 2005 May.
Article in English | MEDLINE | ID: mdl-15711567

ABSTRACT

One of the early features of diabetic retinopathy is the alteration of the blood-retinal barrier (BRB), which may involve the breakdown of endothelial cell tight junctions. The aim of this study was to examine the expression of extracellular proteinases in an animal model of early diabetic retinopathy and to determine their role in the alteration of the BRB. Matrix metalloproteinase (MMP) expression was studied in the retinas of rats with 12 weeks of diabetes. The role of MMPs in regulating tight junction function was investigated in retinal endothelial and pigment epithelial cells by measuring transepithelial electrical resistance (TER). The retinas of diabetic animals demonstrated elevated levels of MMP-2, MMP-9 and MMP-14 messenger RNA. A significant increase in the production of MMP-9 was seen when cells were exposed to high glucose conditions. Both cell types treated with purified MMP-2 or MMP-9 were found to have alterations of tight junction function as shown by decreased TER. Western blot analysis of cell extracts treated with MMP-2 or MMP-9, revealed specific degradation of the tight junction protein, occludin. Results suggest that elevated expression of MMPs in the retina may facilitate an increase in vascular permeability by a mechanism involving proteolytic degradation of the tight junction protein occludin followed by disruption of the overall tight junction complex.


Subject(s)
Blood-Retinal Barrier/enzymology , Diabetes Mellitus, Experimental/enzymology , Diabetic Retinopathy/enzymology , Matrix Metalloproteinases/metabolism , Pigment Epithelium of Eye/enzymology , Animals , Blood-Retinal Barrier/drug effects , Blotting, Western , Cattle , Cell Line , Cell Membrane Permeability , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/pathology , Diabetic Retinopathy/etiology , Diabetic Retinopathy/pathology , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Combinations , Electric Impedance , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Gene Expression Regulation, Enzymologic , Glucose/pharmacology , Humans , Mannose/pharmacology , Matrix Metalloproteinases/genetics , Membrane Proteins/metabolism , Occludin , Pigment Epithelium of Eye/drug effects , Pigment Epithelium of Eye/pathology , Pigment Epithelium of Eye/physiopathology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Tight Junctions/metabolism
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