Abstract 17607: High Content Imaging Assay to Study Sprouting Angiogenesis Demonstrates A Key Role for Macrophage Endothelial Cell Interactions
Background: Two distinct endothelial cell (EC) sub-populations are involved in sprouting angiogenesis. At the forefront of a vessel sprout, "tip cells" guide the sprouting vessel. Behind the tip, "stalk cells" proliferate to elongate and stabilize the vessel branch. Formation of new functional vessels requires a tight coordination between these cellular phenotypes. Here, we developed cell-based assay in a microtiter plate format to facilitate high throughput screens for identifying chemical compounds or genes that regulate sprouting angiogenesis using quantitative high-content imaging.
Methods and results: Mouse EC line (SVEC4-10) were mixed with Matrigel and cultured in 96-well plates (Figure 1). After 4 days of culture, EC sprouts analyzed using automated high-content microscopy (Operetta, Pekin Elmer) and automated image analysis (AngioSys software). Readouts (total tubule length and branching points and mean tubule length) were obtained from images covering the whole well:. Temporal image analysis during 9 days of EC culture showed continuous growth of sprouts and formation of branched network structures. The leading cell of the sprout "tip cell" was characterized by the formation of directional filopodia and the polarized positioning of Golgi apparatus. Incubation with 10 micromoles of DAPT, a Notch signaling inhibitor, resulted in 1.4-fold increase in total tubule length and branching point number (Z' factor =0.36, p=0.03). Next, we investigated the effect of macrophage-EC interaction on sprouting angiogenesis using this model. Co-culture with macrophages (RAW264.7, mouse cell line) decreased the total tubule length and branching point number 1.7-fold and 4-folds, respectively. In contrast, the mean tubule length increased 1.7-fold. These data suggest that macrophages restrict the sprouting behavior of EC and stabilize new endothelial sprouts. This assay can easily be adapted into a high throughput format for chemical or genetic screens.
- © 2012 by American Heart Association, Inc.