Figure 10. VEGF-induced endothelial cell... | Rockefeller University Press

Figure 10.

$Figure 10. VEGF-induced endothelial cell migration requires cadherin endocytosis. (A–E) Wild-type (WT), DEE mutant, and GGG mutant VE-cadherin (VE-cad) was expressed in monolayers of primary human dermal microvascular endothelial cells using an adenoviral transduction system. Infection with an empty adenovirus (EV) was used as a negative control. (A and B) Endothelial monolayers were serum starved for 1 h and then scratched with a pipette tip. 12 h after the scratch, 100 mg/ml VEGF was added to the medium (indicated by arrowheads). Migration of cells into the wound area was tracked over time. Mean distance closed ± SEM (n = 8 wounds per group); *, P < 0.05 compared with empty adenovirus, wild type, and GGG; ◊, P < 0.05 compared with empty adenovirus and wild type only. (C) VE-cadherin expression was measured by Western blotting. Empty arrowhead, endogenous VE-cadherin; filled arrowhead, exogenously expressed VE-cadherin–RFP. (D) Replication was measured by a thymidine analogue incorporation assay. Confluent monolayers were serum starved for 12 h and then either left untreated or treated with VEGF for 6 h, with incubation in EdU during the final hour. After fixation and labeling, the replication rate was estimated by the fraction of infected cells that were EdU positive. Proportion ± standard error (n = 94–103 cells per group). (E) Activation of VEGF signaling was verified by Western blotting for phosphorylated p44/42 MAPK (top) and total p44/42 MAPK (bottom). Cells were serum starved for 12 h and then treated with VEGF or left untreated for 20 min before harvesting. (F and G) Endocytosis of VE-cadherin in endothelial cells migrating into a scratch wound was measured using a fluorescence-based internalization assay. Confluent monolayers of endothelial cells were serum starved for 45 min and then scratched with a pipette tip and either left untreated (control) or treated with VEGF for 45 min. VE-cadherin endocytosis was measured over a 1-h internalization period in cells near to (75 ± 75 µm) or far from (440 ± 75 µm) the wound edge. Means ± SEM (n = 22–32 cells per group). Bars: (A) 100 µm; (F) 20 µm.$

VEGF-induced endothelial cell migration requires cadherin endocytosis. (A–E) Wild-type (WT), DEE mutant, and GGG mutant VE-cadherin (VE-cad) was expressed in monolayers of primary human dermal microvascular endothelial cells using an adenoviral transduction system. Infection with an empty adenovirus (EV) was used as a negative control. (A and B) Endothelial monolayers were serum starved for 1 h and then scratched with a pipette tip. 12 h after the scratch, 100 mg/ml VEGF was added to the medium (indicated by arrowheads). Migration of cells into the wound area was tracked over time. Mean distance closed ± SEM (n = 8 wounds per group); *, P < 0.05 compared with empty adenovirus, wild type, and GGG; ^◊, P < 0.05 compared with empty adenovirus and wild type only. (C) VE-cadherin expression was measured by Western blotting. Empty arrowhead, endogenous VE-cadherin; filled arrowhead, exogenously expressed VE-cadherin–RFP. (D) Replication was measured by a thymidine analogue incorporation assay. Confluent monolayers were serum starved for 12 h and then either left untreated or treated with VEGF for 6 h, with incubation in EdU during the final hour. After fixation and labeling, the replication rate was estimated by the fraction of infected cells that were EdU positive. Proportion ± standard error (n = 94–103 cells per group). (E) Activation of VEGF signaling was verified by Western blotting for phosphorylated p44/42 MAPK (top) and total p44/42 MAPK (bottom). Cells were serum starved for 12 h and then treated with VEGF or left untreated for 20 min before harvesting. (F and G) Endocytosis of VE-cadherin in endothelial cells migrating into a scratch wound was measured using a fluorescence-based internalization assay. Confluent monolayers of endothelial cells were serum starved for 45 min and then scratched with a pipette tip and either left untreated (control) or treated with VEGF for 45 min. VE-cadherin endocytosis was measured over a 1-h internalization period in cells near to (75 ± 75 µm) or far from (440 ± 75 µm) the wound edge. Means ± SEM (n = 22–32 cells per group). Bars: (A) 100 µm; (F) 20 µm.