Reduced Pericyte Adhesion and Death on Advanced Glycation Endproduct (Age)–Modified Substrate

Abstract

Abstract: : Purpose: An early pathophysiological hallmark of diabetic retinopathy is the premature death of retinal microvascular perictyes (RMP). Diabetes–mediated modifications of the basement membrane (BM) via advanced glycation reactions may play a significant role in promoting RMP dysfunction and death by depleting important cell recognition and survival motifs. In the current study we have employed an in vitro model of AGE–modified BM to evaluate RMP interaction with "diabetic–like" substrates and the pathophysiologic role they play in diabetic retinopathy. Methods: AGE–BM was prepared and fully characterised according to established protocols (Stitt et al. Diabetologia 2004:47:1735–1746). Primary cultures of bovine RMPs were isolated and propagated on native BM or AGE–modified BM. RMP attachment and spreading on these BM preparations was determined morphologically, whilst caspase 3 activity and mitochondrial depolarisation assays were used to evaluate cell dysfunction leading to premature death. The effect of supplementation of the AGE–modified substrata with exogenous RGD and YIGSR cell recognition peptide motifs on RMP adhesion and survival was also assessed. Results: RMPs seeded onto AGE–BM showed a significant (p<0.01–0.001) dose dependent reduction in adhesive capacity compared to cells exposed to unmodified controls. Caspase 3 activity increased in cells exposed to AGE–modified matrix whilst there was a significant reduction in red:green fluorescence ratio of JC–1 stained RMPs indicative of mitochondrial membrane polarization and early apoptosis ( p=0.0022, p<0.01). Supplementing AGE–modified matrix with YIGSR peptide (5mM) prior to cell seeding enhanced RMP adhesion, however supplementation with RGD peptide (5mM) was inhibitory. Neither peptide had an effect of RMP spreading individually, although highly modified AGE substrata supplemented with both peptides enhanced RMP spreading capability. Conclusions: AGE modification of BM proteins significantly impaired RMP attachment and survival, via caspase dependent cell death mechanisms. Furthermore, supplementation of AGE–modified matrices with exogenous YIGSR and RGD peptides, replacing AGE–mediated depletion of cell recognition motifs, reversed some of the dysfunctional effects associated with AGE exposure.

Keywords: cell adhesions/cell junctions • cell death/apoptosis • diabetic retinopathy