Matrix Stiffness-Mediated DNA Methylation in Endothelial Cells.

Purpose: Altered tissue mechanics is a prominent feature of many pathological conditions including cancer. As such, much work has been dedicated to understanding how mechanical features of tissues contribute to pathogenesis. Interestingly, previous work has demonstrated that the tumor vasculature acquires pathological features in part due to enhanced tumor stiffening. To further understand how matrix mechanics may be translated into altered cell behavior and ultimately affect tumor vasculature function, we have investigated the effects of substrate stiffening on endothelial epigenetics. Specifically, we have focused on DNA methylation as recent work indicates DNA methylation in endothelial cells can contribute to aberrant behavior in a range of pathological conditions. Methods: Human umbilical vein endothelial cells (HUVECs) were seeded on stiff and compliant collagen-coated polyacrylamide gels and allowed to form monolayers over 5 days. DNA methylation was assessed via 5-methylcytosine ELISA assays and immunofluorescent staining. Gene expression was assessed via qPCR on RNA isolated from HUVECs seeded on collagen-coated polyacrylamide gels of varying stiffness. Results: Our work demonstrates that endothelial cells cultured on stiffer substrates exhibit lower levels of global DNA methylation relative to endothelial cells cultured on more compliant substrates. Interestingly, gene expression and DNA methylation dynamics suggest stiffness-mediated gene expression may play a role in establishing or maintaining differential DNA methylation levels in addition to enzyme activity. Additionally, we found that the process of passaging induced higher levels of global DNA methylation. Conclusions: Altogether, our results underscore the importance of considering cell culture substrate mechanics to preserve the epigenetic integrity of primary cells and obtain analyses that recapitulate the primary environment. Furthermore, these results serve as an important launching point for further work studying the intersection tissue mechanics and epigenetics under pathological conditions.