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SJ Hughes, TA Gardiner, O Lynch, AW Stitt; Age-modified Sub-cellular Matrix Modulates Retinal Pericyte Dysfunction and Death: Implications for Diabetic Retinopathy . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1351.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Advanced glycation end-products (AGEs) accumulate in the retinal capillaries during diabetes where they may have many deleterious effects. Within these vessels, AGE-crosslinking of basement membrane (BM) components may result in compromised pericyte-matrix interactions and progressive depletion of signalling from important survival factors such as platelet-derived growth factor (PDGF). We determined if AGE-modified BM (AGE-BM) could alter retinal pericyte function and survival in vitro and if replenishment of PDGF could protect against premature death. Methods: Matrigel (BM extract) was incubated with glycoaldehyde (10-500mM) for 4 hours to produce AGE-BM of defined lysine depletion and crosslinking properties. Bovine retinal pericytes (BRPs) were then grown on increasingly AGE-modified BM and the ability of the cells to spread was quantified. Filamentous actin was localised with fluorescently-labelled phalloidin and visualised using confocal microscopy. BRP apoptotic death was quantified in cells growing on AGE-BM. Apoptosis was also assessed in BRPs after addition of PDGF-AA or PDGF-BB to the modified matrix. Phosphorylation of the downstream kinase Akt was also investigated in AGE-exposed BRPs using immunoprecipitation and western blotting of whole cell homogenates. Results: BRPS grown on AGE-BM showed a markedly reduced spreading capacity with a concomitant disruption of the actin cytoskeleton when compared to native BM. BRPs exposed to AGE-BM also demonstrated a step-wise increase in apoptotic death when compared to controls (P<0.001 at highest degree of modification) and this correlated with decreased phosphorylation of Akt. Supplementation with PDGF-AA or PDGF-BB (0.25µg/ml) significantly protected BRPs against apoptotic death in a dose-dependent manner (P<0.001). PDGF-AA was slightly less effective than PDGF-BB in the prevention of apoptotic death in BRPs. Conclusion: BRPs grown on AGE-BM show a range of dysfunctional effects. After prolonged exposure to AGE-BM, BRPs showed markedly increased apoptosis, that which could be prevented by the addition of PDGF to the underlying BM. These findings may have implications for pericyte dysfunction and death during diabetic retinopathy.
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