An extracellular matrix protein microarray to study endothelial to mesenchymal transformation in mitral valve endothelial cells

In the developing heart, a subset of endothelial cells (ECs) undergo a process of endothelial to mesenchymal transformation (EMT) that leads to the remodeling of the cardiac cushions and the formation of the cardiac valves. Understanding how microenvironmental cues regulate EMT has important implication for the treatment of congenital heart valve diseases and for heart valve tissue engineering. Here a set of extracellular matrix (ECM) proteins and growth factors (GF) were tested to determine the extent of regulation of EMT in adult ovine mitral valve endothelial cells (MVECs). Traditional cell culture dishes present limitations on large scale screening of cells-microenvironments interactions. To address this problem, a high throughput cellular microarray platform was developed. Using a contact printer, 84 different combinations of ECM proteins and GF were microarrayed on a modified glass slide. The slides were seeded with MVECs and immunostained against EMT maker alpha smooth muscle actin ([alpha]-SMA). Fluorescence images of each spot of the microarray were taken with a microscope equipped with an automated stage. These images were analyzed with a custom image processing routine developed with CellProfiler to assess the percentage of cells expressing [alpha]-SMA on each ECM combination.