Advanced glycation of the arg-gly-asp (RGD) tripeptide motif modulates retinal microvascular endothelial cell dysfunction

Molecular vision

McDonald, Denise M.¹  Gardiner, Tom A.¹  Bhatwadekar, Ashay¹  Coleman, Gary¹  Stitt, Alan W.¹ 

DOI  :  

学科分类：生物化学/生物物理

来源: Molecular Vision

【 摘 要 】

Purpose: Advanced glycation endproduct(AGE) formation on the basement membrane of retinal capillaries hasbeen previously described but the impact of these adducts on capillaryendothelial cell function vascular repair remains uncertain. Thisinvestigation has evaluated retinal microvascular endothelial cells(RMECs) growing on AGE-modified fibronectin (FN) and determined howthis has an impact on cell-substrate interactions and downstreamoxidative responses and cell survival. Methods: RMECs were grown onmethylglyoxal-modified FN (AGE-FN) or native FN as a control. RMECattachment and spreading was quantified. In a separate treatment, theAGE-FN substrate had Arg-Gly-Asp-Ser (RGDS) or scrambled peptide addedbefore seeding. Phosphorylation of focal adhesion kinase (FAK) and α5β1integrin localization was assessed and apoptosis evaluated. In a subsetof RMECs that remained attached to the AGE-FN substrate, the productionof superoxide (O2-) was assayed usingdihydroethidium (DHE) fluorescence or lucigenin, in the presence orabsence of NADPH. The specificity of the O2-assays was confirmed by inhibition in the presence ofpolyethylene-glycol-superoxide dismutase (PEG-SOD). AGE-mediatedchanges to mRNAs encoding key basement membrane proteins and regulatoryenzymes were investigated using real-time RT–PCR. Results: AGE-FN reduced RMEC attachmentand spreading when compared to FN controls (p<0.001). RGDS peptideenhanced cell attachment on AGE-FN (p<0.001), while the scrambledpeptide had no effect. FAK phosphorylation in AGE-exposed RMECs wasreduced in a time-dependent fashion, while α5β1integrin-immunoreactivity became focal at the basal membrane.AGE-exposure induced apoptosis, a response significantly prevented byRGDS peptide. AGE-exposure caused a significant increase in basal O2-and NADPH-stimulated production by RMECs (p<0.01), while AGE-FN alsoincreased basement membrane associated mRNA expression (p<0.05). Conclusions: AGE substrate modificationsimpair the function of retinal capillary endothelium and theirreparative potential in response to diabetes-related insults.Arginine-specific modifications alter vital endothelial cellinteractions with the substrate. This phenomenon could play animportant role in dysfunction and nonperfusion of retinal capillariesduring diabetes.

【 授权许可】

Unknown