Examination of the leading edge revealed the formation of endothelial leader cells with an aggressive phenotype at the protrusion tip (Fig. converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters. The endothelium forms the inner lining of blood vessels and plays essential roles in vascular biology1. It provides a functional barrier for retaining circulating blood, regulating blood-tissue exchange, recruiting blood cells, and controlling vascular tone. The integrity of the endothelium plays an important role in its physiological Rabbit polyclonal to Nucleostemin function. Upon mechanical injury, e.g., iatrogenic operations, endothelial cells migrate to heal the Ciprofloxacin hydrochloride hydrate wound autonomously and maintain the functions of the vasculature. Endothelial cells crawl collectively atop the wound bed to restore the integrity of the endothelium. The migration of endothelial cells is modularly controlled to regulate cell motility, directed migration, cell-cell coordination, and cell density2,3. Calcium signaling components, such as phospholipase C, stromal interaction molecule 1 and diacylglycerol, are polarized at the leading edge to promote persistent forward migration4. Previous studies of collective cell migration, however, were performed primarily using epithelial cells. A better understanding of endothelial collective migration is essential for the development of novel therapeutics and tissue engineering approaches to treat endothelial dysfunction and vascular diseases. The formation of leader cells are observed during epithelial wound closure5,6. In particular, leader cells with an aggressive phenotype emerge near the wound boundary and mechanically interact with follower cells to form multicellular migrating clusters7. Mechanical force and the Rho signaling pathway are known to modulate the formation of leader cells8,9,10,11. Inhibition of Rho signaling with Y-27632 increases the leader cell density at the leading edge. Recently, Notch1-Dll4 lateral inhibition is also shown to regulate leader cell formation during epithelial collective migration12. Despite its importance in vascular biology, the regulation of endothelial leader cells under various physiological conditions, such as different wound sizes and shapes, remain poorly understood. More importantly, the functional relationship between leader cells and follower cells in the migrating clusters has not been explored due to the challenge of adjusting the leader cell density systematically. The ability to non-invasively modulate Ciprofloxacin hydrochloride hydrate the leader cell density will be invaluable for deciphering the functions of leader cells. In this study, we develop a plasma lithography modulated would healing assay to study the regulation and function of leader cells. Collective migration of human umbilical vein endothelial cells (HUVEC), which is an established model system for human endothelial cells, is studied on geometric patterns created by surface plasma treatment13,14,15,16,17. In the assay, the geometry of the cell monolayer is controlled by spatially patterning the hydrophobicity of the substrate and collective migration is induced by removing a physical Ciprofloxacin hydrochloride hydrate blocker to create a cell free region in the pattern18. The plasma lithography technique has been previously demonstrated for investigating several biological systems19,20,21. The formation of leader cells and the migration rate of the monolayer are investigated in rectangular patterns of various dimensions. Converging, diverging and competing patterns are also designed to perturb the leader cell density non-invasively. The Ciprofloxacin hydrochloride hydrate leader cells and follower cells in the migrating cluster are characterized by immunostaining, time-lapse microscopy and particle image velocimetry (PIV). The influences of the leader cell density on the migrating cluster and the overall migration rate of the monolayer are investigated to elucidate the functions of leader cells. Results The density of migrating clusters is independent of the pattern width A plasma lithography modulated wound healing assay was developed to study endothelial collective migration (Fig. 1A and supplementary Fig. S1A). In this assay, a polydimethylsiloxane (PDMS) mold was applied to shield specific regions of a polystyrene substrate from air plasma treatment. Selective plasma treatment created hydrophilic patterns that promote cell adhesion. A cell free region for cell migration was generated by placing a PDMS blocker onto the appropriate location of the polystyrene dish before cell seeding18. Upon the release of the blocker, the cell monolayer migrated toward the cell free region. Consistent with other injury-free assays22,23,24, the release of contact inhibition was sufficient to induce collective migration on the patterns (supplementary Fig. S1B). The technique allowed us to.