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A. Bhatwadekar, T.A. Gardiner, T.M. Curtis, G. Li, A.W. Stitt; Advanced Glycation Impairs Endothelial Progenitor Cell Attachment and Repair in a New Model of Retinal Vascular Endothelial Injury . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3524.
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© ARVO (1962-2015); The Authors (2016-present)
Vascular repair by marrow–derived endothelial progenitor cells (EPCs) is impaired in diabetes although it is unknown if such dysfunction occurs in the diabetic retina. We hypothesised that accumulation of advanced glycation endproducts (AGEs) on capillary basement membranes (BMs), as occurs during diabetic retinopathy, could hamper EPCs vasoreparative function in this specialised microvascular bed. Using a novel in vitro model, whereby delineated regions of apoptotic death were induced in monolayers of retinal microvascular endothelial cells (RMECs), we assessed the potential for AGE–modification of BM to impair EPC function and incorporation into RMEC monolayers.
EPCs were purified from human peripheral blood and characterised by staining with acLDL–DiI, UEA–1, CD31, and VEGFR2. Native fibronectin (FN) was used as a control while methylglyoxal (MGO)–modified FN (AGE–FN) was used as a "diabetic–like" substrate in which AGE adducts CML and argpyrimidine were quantified. EPC interactions with AGE–FN were assessed for attachment and spreading at 3 and 6 hrs respectively. EPC migration in response to SDF–1 was quantified using Dunn’s chemotaxis assay. RMEC monolayers were treated with verteporfin (1µg/ml) which was subsequently activated using focused red light causing localised apoptosis. The ability of exogenous EPCs to "endothelialise" these wounds was then assessed.
EPC attachment was reduced to 25% (p<0.05) while spreading was reduced to 32% (p<0.01) of control levels on AGE–FN. Preincubation of EPCs with RGD peptide reduced EPC attachment to 52 % of control levels (p<0.05) while modification of same with MGO eliminated its inhibitory effect. EPC migration was reduced over AGE–FN with no specific trajectory along SDF–1 gradient which was evident in controls. RMEC monolayers wounded with verteporfin exhibited enhanced expression of ICAM, VCAM at the edge of the denuded area. Addition of DiI labelled EPCs to wounded RMEC monolayers resulted in specific targeting, with 5 times more EPCs near the wound site as compared to untreated regions (p<0.001). AGE–FN caused 3 fold reductions in EPCs incorporation into RMEC monolayers which was dependent on the degree of AGE modification.
This study suggests that EPCs could play a hitherto unrecognised role in retinal capillary endothelial repair. AGE modification of BM protein decreases EPC vasoreparative function in vitro. This has important implications for progressive vasodgeneration during diabetic retinopathy.
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