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Proliferation assays; Confluent and sparse HUVEC cultures

Immunofluorescence analysis of c-met and VE-cadherin expression in wound healing assay

An in vitro wound healing assay (ie, the ability to fill in artificial gaps created in cell monolayers) requires both cell growth and cell migration. We used an in vitro wound healing assay to investigate whether the HGF receptor was expressed differently in a normal and a repairing endothelium. The surface of a confluent HUVEC monolayer was scratched and we followed its healing during incubation in regular medium. shows an area in which HUVECs were engaged in the process of repair. The expression of c-met and VE-cadherin in this area was monitored by use of double-staining immunofluorescence analysis. During the first 12 hours after scratching, single HUVECs detached from the border of the confluent monolayer and moved into the damaged area These single cells were not stained with anti–VE-cadherin antibody but were stained with the anti–c-met antibody. The neighboring undamaged region was not labeled with anti–c-met antibody. Interestingly, VE-cadherin immunolabeling of this region was also drastically reduced. More cells in the moving edge were labeled with anti–c-met antibody after 12 more hours of incubation when most of the cells were engaged in the healing process. These moving cells were still not labeled with the anti–VE-cadherin antibody, whereas VE-cadherin immunostaining was reinforced in the adjacent confluent monolayer. After 48 hours of incubation, when migrating cells had completely filled the free space, c-met immunolabeling disappeared and VE-cadherin immunolabeling was restored to a level comparable to that in the undamaged confluent monolayer. Thus, c-met expression is up-regulated during the repair of the endothelium.