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double-staining experiments

We could hardly detect c-met mRNA in confluent HUVECs. However, the amount of c-met mRNA increased dramatically when the cells were switched to low-density conditions or during the formation of capillary-like structures in the 3-D collagen gel In contrast, high levels of VE-cadherin mRNA were detected in confluent HUVECs, whereas substantially lower levels were observed in sparse conditions. The amounts of VE-cadherin mRNA increased when HUVECs were cultured in a 3-D collagen gel.

For comparison, we observed no changes in the amount of β-actin mRNA in any of the conditions

Effect of cell density on the mitogenic response of HUVECs to HGF

To determine the functional consequence of the density-dependent changes in c-met expression, we examined the effect of HGF on the proliferation of HUVECs harvested from confluent monolayers and from a duplicate culture of the same cells that had been replated at a lower density after the last feeding ( [3H]Thymidine incorporation experiments showed that HGF stimulated the proliferation in a dose-dependent manner in both confluent and sparse HUVECs. Moreover, the treatment with HGF led to an expected increased stimulation of DNA synthesis in sparse HUVECs, compared to that in confluent HUVECs. The difference between the 2 growth conditions was greatest at the lowest HGF concentration (1 ng/mL). In these conditions, the effect of HGF was nearly 30-fold greater in sparse cells than in confluent cells (42% and 1.4% stimulation, respectively). For comparison, the effect was only 2.6-fold higher in sparse HUVECs than in confluent HUVECs when cells were treated with 25 ng/mL HGF (227% and 86% stimulation, respectively)