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N Frizzell, TA Gardiner, CB T McMullen, SR Thorpe, V Brown, AW Stitt; Basement Membrane-immobilised Advanced Glycation Endproducts Induce Capillary Endothelial Dysfunction And Apoptosis . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1350.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:During diabetes elevated levels of advanced glycation endproducts (AGEs) accumulate in the retinal capillaries and their basement membranes (BM). AGE-crosslinking of BM components may result in compromised cell-matrix interactions. The current investigation examined responses of retinal microvascular endothelial cells (RMECs) growing on AGE-modifed substrate in vitro. In particular we studied the regulation of ICAM-1 and NF-kB, two proteins known to be upregulated during retinal leukostasis. Methods:BM extracts were incubated with glycoaldehyde (10-500mM) for 4 hours to produce AGE-modified BM proteins (AGE-BM). The extent of AGE modification and crosslinking was measured by GC/MS and amino acid analysis. RMECs were isolated and cultured on native or AGE-BM for various times. Phase contrast microscopy was used to visualise RMEC adherence and the number of apoptotic cells were quantified. RMEC spreading was analysed by staining F-actin microfilaments with fluorescently-labelled phalloidin followed by confocal microscopy. The nuclear translocation of NF-kB component p65 was demonstrated using confocal microscopy and an ELISA based assay. The surface expression of ICAM-1 was determined by using flow cytometry. Results:Glycoaldehyde resulted in a stepwise increase in the AGE carboxymethly lysine (CML) formation (mmol/mol lysine) on AGE-BM and amino acid analysis showed up to 50% depletion of lysine residues. RMECs grown on AGE-BM demonstrated greater levels of apoptosis compared to controls (23±4.23 vs. 97±7.41 p<0.05). Cells exposed to AGE-BM also showed disrupted F-actin concomitant with a decrease in normal cell spreading in comparison to RMECs grown on native BM extract. AGE-BM also induced nuclear translocation of NF-kB p65 and enhanced DNA binding of p65 (0.35 OD vs. 0.19 OD, p<0.05). A stepwise up-regulation of ICAM-1 on the RMEC surface was demonstrated after 16hrs exposure to AGE-ECM. Conclusion:Exposure to AGE-BM can induce a range of dysfunctional effects in RMECs. This is a highly relevant route for AGE-exposure, and the effects demonstrated may have important implications for capillary endothelial cell dysfunction during diabetic retinopathy.
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