645 Study of the impact of age-related alterations of the extracellular matrix on the organization of the network of endothelial cells using an in vitro cell co-culture model

Abstract

With age, dermis loose its elasticity. Its main component, Collagen, undergo decreased synthesis and degradation. Elastin and Fibronectin are also impacted. In addition, the extrafibrillar matrix shows decreased level of glycosaminoglycans, one of its abundant components. Fibroblasts, key players in the production and organisation of the extracellular matrix (ECM), present reduced growth and modified secretion patterns. Another aspect of ageing is the alteration of the microcirculation network that limits supply of nutrient to the dermis. To study the impact of ECM on the organisation of the microcirculation network, we developed an in vitro cell model associating fibroblasts and endothelial cells. We cultured fibroblast from a young (23 years-old) or an old (71 years-old) donor. At day 7, we first used RT-qPCR, to compare the expression levels of selected genes related to ECM. We also added endothelial cells on top of the fibroblasts, and after 5 additional days of co-culture, studied the quality of the network of endothelial cell by immunofluorescent labelling of CD31. Comparative genes’ expression analysis reveals that fibroblasts from an old donor show decreased expression of genes involved in the synthesis or maintenance of the ECM (VCAN, GPC4) or with its extrafibrillar components (CD44, HAS2), while the expression of genes associated with ECM degradation is increased (MMP1 and MMP4). Immunofluorescent labelling of CD31 shows that the network of endothelial cells is of good quality when co-culture is performed with fibroblasts from a young donor. This quality significantly decreases if co-culture is performed with fibroblast from an old donor. Therefore, the matrix environment from an aged donor negatively impacts the development of the network of endothelial cells. This effect can be associated with the under-expression of ECM components but also with extrafibrillar elements.