Abstract
PURPOSE: A critical event in the pathogenesis of diabetic retinopathy is the inappropriate adherence of leukocytes to the retinal capillaries. Advanced glycation end-products (AGEs) are known to play a role in chronic inflammatory processes, and the authors postulated that these adducts may play a role in promoting pathogenic increases in proinflammatory pathways within the retinal microvasculature. METHODS: Retinal microvascular endothelial cells (RMECs) were treated with glycoaldehyde-modified albumin (AGE-Alb) or unmodified albumin (Alb). NFkappaB DNA binding was measured by electromobility shift assay (EMSA) and quantified with an ELISA: In addition, the effect of AGEs on leukocyte adhesion to endothelial cell monolayers was investigated. Further studies were performed in an attempt to confirm that this was AGE-induced adhesion by co-incubation of AGE-treated cells with soluble receptor for AGE (sRAGE). Parallel in vivo studies of nondiabetic mice assessed the effect of intraperitoneal delivery of AGE-Alb on ICAM-1 mRNA expression, NFkappaB DNA-binding activity, leukostasis, and blood-retinal barrier breakdown. RESULTS: Treatment with AGE-Alb significantly enhanced the DNA-binding activity of NFkappaB (P = 0.0045) in retinal endothelial cells (RMECs) and increased the adhesion of leukocytes to RMEC monolayers (P = 0.04). The latter was significantly reduced by co-incubation with sRAGE (P <0.01). Mice infused with AGE-Alb demonstrated a 1.8-fold increase in ICAM-1 mRNA when compared with control animals (P <0.001, n = 20) as early as 48 hours, and this response remained for 7 days of treatment. Quantification of retinal NFkappaB demonstrated a threefold increase with AGE-Alb infusion in comparison to control levels (AGE Alb versus Alb, 0.23 vs. 0.076, P <0.001, n = 10 mice). AGE-Alb treatment of mice also caused a significant increase in leukostasis in the retina (AGE-Alb versus Alb, 6.89 vs. 2.53, n = 12, P <0.05) and a statistically significant increase in breakdown of the blood-retinal barrier (AGE Alb versus Alb, 8.2 vs. 1.6 n = 10, P <0.001). CONCLUSIONS: AGEs caused upregulation of NFkappaB in the retinal microvascular endothelium and an AGE-specific increase in leukocyte adhesion in vitro was also observed. In addition, increased leukocyte adherence in vivo was demonstrated that was accompanied by blood-retinal barrier dysfunction. These findings add further evidence to the thinking that AGEs may play an important role in the pathogenesis of diabetic retinopathy.
Original language | English |
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Pages (from-to) | 4457-4464 |
Number of pages | 8 |
Journal | Investigative Ophthalmology and Visual Science |
Volume | 44 |
Issue number | 10 |
Publication status | Published - 01 Oct 2003 |
Bibliographical note
LR: 20100324; GR: EY11627/EY/NEI NIH HHS/United States; GR: EY12611/EY/NEI NIH HHS/United States; JID: 7703701; 0 (Glycosylation End Products, Advanced); 0 (NF-kappa B); 0 (RNA, Messenger); 126547-89-5 (Intercellular Adhesion Molecule-1); 9007-49-2 (DNA); ppublishKeywords
- Animals
- Blood-Retinal Barrier/drug effects
- Capillaries
- Capillary Permeability
- Cattle
- Cell Adhesion/drug effects
- DNA/metabolism
- Electrophoretic Mobility Shift Assay
- Endothelium, Vascular/metabolism
- Enzyme-Linked Immunosorbent Assay
- Glycosylation End Products, Advanced/pharmacology
- Humans
- Intercellular Adhesion Molecule-1/genetics
- Male
- Mice
- Mice, Inbred C57BL
- NF-kappa B/metabolism
- Neutrophils/physiology
- RNA, Messenger/metabolism
- Retinal Vessels/metabolism
- Reverse Transcriptase Polymerase Chain Reaction
- Up-Regulation