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Advanced glycation of the Arg-Gly-Asp (RGD) tripeptide motif modulates retinal microvascular endothelial cell dysfunction

PURPOSE: Advanced glycation endproduct (AGE) formation on the basement membrane of retinal capillaries has been previously described but the impact of these adducts on capillary endothelial cell function vascular repair remains uncertain. This investigation has evaluated retinal microvascular endoth...

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Autores principales: McDonald, Denise M., Coleman, Gary, Bhatwadekar, Ashay, Gardiner, Tom A., Stitt, Alan W.
Formato: Texto
Lenguaje:English
Publicado: Molecular Vision 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2722710/
https://www.ncbi.nlm.nih.gov/pubmed/19668595
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author McDonald, Denise M.
Coleman, Gary
Bhatwadekar, Ashay
Gardiner, Tom A.
Stitt, Alan W.
author_facet McDonald, Denise M.
Coleman, Gary
Bhatwadekar, Ashay
Gardiner, Tom A.
Stitt, Alan W.
author_sort McDonald, Denise M.
collection PubMed
description PURPOSE: Advanced glycation endproduct (AGE) formation on the basement membrane of retinal capillaries has been previously described but the impact of these adducts on capillary endothelial cell function vascular repair remains uncertain. This investigation has evaluated retinal microvascular endothelial cells (RMECs) growing on AGE-modified fibronectin (FN) and determined how this has an impact on cell-substrate interactions and downstream oxidative responses and cell survival. METHODS: RMECs were grown on methylglyoxal-modified FN (AGE-FN) or native FN as a control. RMEC attachment and spreading was quantified. In a separate treatment, the AGE-FN substrate had Arg-Gly-Asp-Ser (RGDS) or scrambled peptide added before seeding. Phosphorylation of focal adhesion kinase (FAK) and α5β1 integrin localization was assessed and apoptosis evaluated. In a subset of RMECs that remained attached to the AGE-FN substrate, the production of superoxide (O(2)(-)) was assayed using dihydroethidium (DHE) fluorescence or lucigenin, in the presence or absence of NADPH. The specificity of the O(2)(-) assays was confirmed by inhibition in the presence of polyethylene-glycol-superoxide dismutase (PEG-SOD). AGE-mediated changes to mRNAs encoding key basement membrane proteins and regulatory enzymes were investigated using real-time RT–PCR. RESULTS: AGE-FN reduced RMEC attachment and spreading when compared to FN controls (p<0.001). RGDS peptide enhanced cell attachment on AGE-FN (p<0.001), while the scrambled peptide had no effect. FAK phosphorylation in AGE-exposed RMECs was reduced in a time-dependent fashion, while α5β1 integrin-immunoreactivity became focal at the basal membrane. AGE-exposure induced apoptosis, a response significantly prevented by RGDS peptide. AGE-exposure caused a significant increase in basal O(2)(-) and NADPH-stimulated production by RMECs (p<0.01), while AGE-FN also increased basement membrane associated mRNA expression (p<0.05). CONCLUSIONS: AGE substrate modifications impair the function of retinal capillary endothelium and their reparative potential in response to diabetes-related insults. Arginine-specific modifications alter vital endothelial cell interactions with the substrate. This phenomenon could play an important role in dysfunction and nonperfusion of retinal capillaries during diabetes.
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spelling pubmed-27227102009-08-07 Advanced glycation of the Arg-Gly-Asp (RGD) tripeptide motif modulates retinal microvascular endothelial cell dysfunction McDonald, Denise M. Coleman, Gary Bhatwadekar, Ashay Gardiner, Tom A. Stitt, Alan W. Mol Vis Research Article PURPOSE: Advanced glycation endproduct (AGE) formation on the basement membrane of retinal capillaries has been previously described but the impact of these adducts on capillary endothelial cell function vascular repair remains uncertain. This investigation has evaluated retinal microvascular endothelial cells (RMECs) growing on AGE-modified fibronectin (FN) and determined how this has an impact on cell-substrate interactions and downstream oxidative responses and cell survival. METHODS: RMECs were grown on methylglyoxal-modified FN (AGE-FN) or native FN as a control. RMEC attachment and spreading was quantified. In a separate treatment, the AGE-FN substrate had Arg-Gly-Asp-Ser (RGDS) or scrambled peptide added before seeding. Phosphorylation of focal adhesion kinase (FAK) and α5β1 integrin localization was assessed and apoptosis evaluated. In a subset of RMECs that remained attached to the AGE-FN substrate, the production of superoxide (O(2)(-)) was assayed using dihydroethidium (DHE) fluorescence or lucigenin, in the presence or absence of NADPH. The specificity of the O(2)(-) assays was confirmed by inhibition in the presence of polyethylene-glycol-superoxide dismutase (PEG-SOD). AGE-mediated changes to mRNAs encoding key basement membrane proteins and regulatory enzymes were investigated using real-time RT–PCR. RESULTS: AGE-FN reduced RMEC attachment and spreading when compared to FN controls (p<0.001). RGDS peptide enhanced cell attachment on AGE-FN (p<0.001), while the scrambled peptide had no effect. FAK phosphorylation in AGE-exposed RMECs was reduced in a time-dependent fashion, while α5β1 integrin-immunoreactivity became focal at the basal membrane. AGE-exposure induced apoptosis, a response significantly prevented by RGDS peptide. AGE-exposure caused a significant increase in basal O(2)(-) and NADPH-stimulated production by RMECs (p<0.01), while AGE-FN also increased basement membrane associated mRNA expression (p<0.05). CONCLUSIONS: AGE substrate modifications impair the function of retinal capillary endothelium and their reparative potential in response to diabetes-related insults. Arginine-specific modifications alter vital endothelial cell interactions with the substrate. This phenomenon could play an important role in dysfunction and nonperfusion of retinal capillaries during diabetes. Molecular Vision 2009-08-05 /pmc/articles/PMC2722710/ /pubmed/19668595 Text en http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
McDonald, Denise M.
Coleman, Gary
Bhatwadekar, Ashay
Gardiner, Tom A.
Stitt, Alan W.
Advanced glycation of the Arg-Gly-Asp (RGD) tripeptide motif modulates retinal microvascular endothelial cell dysfunction
title Advanced glycation of the Arg-Gly-Asp (RGD) tripeptide motif modulates retinal microvascular endothelial cell dysfunction
title_full Advanced glycation of the Arg-Gly-Asp (RGD) tripeptide motif modulates retinal microvascular endothelial cell dysfunction
title_fullStr Advanced glycation of the Arg-Gly-Asp (RGD) tripeptide motif modulates retinal microvascular endothelial cell dysfunction
title_full_unstemmed Advanced glycation of the Arg-Gly-Asp (RGD) tripeptide motif modulates retinal microvascular endothelial cell dysfunction
title_short Advanced glycation of the Arg-Gly-Asp (RGD) tripeptide motif modulates retinal microvascular endothelial cell dysfunction
title_sort advanced glycation of the arg-gly-asp (rgd) tripeptide motif modulates retinal microvascular endothelial cell dysfunction
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2722710/
https://www.ncbi.nlm.nih.gov/pubmed/19668595
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