AGE/RAGE and PKC‐zeta interplay in 3D matrix mediated fibroblast differentiation

Abstract

This purpose of this study was to determine whether increases in advanced glycation endproducts (AGEs) in a 3D diabetic collagen matrix will differentiate wild type (WT) cardiac fibroblasts to a profibrotic phenotype. 3D collagen matrices were prepared from collagen extracts from non‐diabetic (Db/db) and leptin receptor deficient, diabetic (db/db) mouse tails. Primary cardiac fibroblasts isolated from WT and AGE receptor deficient (R−/−) mice which were seeded onto both Db/db and db/db 3D collagen matrices for 7 days (chronic exposure). In addition, on day 6, these cells were then treated with inhibitors UO126 (ERK 1/2 inhibitor; 10μM) and PKC‐zeta Pseudosubstrate (PKC‐zeta inhibitor; 1μg/ml) and with a RAGE ligand‐ glycated albumin (AGE‐BSA; 0.5mg/ml) to induce RAGE activation. Chronically exposed R−/− cells were unchanged, however WT cells exhibited functional and phenotypical markers for fibroblast differentiation, such as increased alpha‐smooth muscle actin and RAGE expression. Additionally, blockade of ERK1/2 and PKC‐zeta restored WT expression to non‐diabetic levels. Therefore, chronic exposure to AGE‐crosslinked diabetic ECM resulted in phenotypic alterations in WT fibroblasts. These changes were mediated through AGE/RAGE interactions leading to a profibrotic cell phenotype.