Figure 6
Figure 6. Dll4-FL inhibits EC migration. (A) HUVECs were transfected with expression vectors for Dll4-Fl or vector alone and sorted for transfected cells. Confluent cultures of HUVECs were scraped with a plastic Pasteur pipette to produce a 3-mm–wide cell-free zone in the monolayer. The ability of the cells to migrate and close the wound was assessed over 18 hours. Dll4-Fl inhibited the migration of ECs even in the presence of VEGF, while exogenous sDll4 abolished this inhibition. (B) Endothelial cell migration into the cell free zone was quantitated using Bioquant Image Analysis (mean ± SEM from triplicate wells in 2 repetition experiments). *P < .05 when Dll4-Fl is compared with vector alone treated either with VEGF or sDll4-His. Dll4-Fl inhibits tubule formation in vitro. Photomicrographs were taken with a Nikon Plan Fluor, 0.17, 4×/0.12 NA objective and 10× eyepiece and processed with Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD). (C) HUVECs were transfected with expression vectors for Dll4-Fl or vector alone and sorted cells were cultured on standard Matrigel in growth factor–deficient conditions in triplicates in 2 independent experiments with either sDll4 or VEGF for 18 hours. Shown are representative pictures from triplicate wells repeated twice. Quantitative analysis for tube length and the number of junctions in various groups is presented. *P < .05 compared with no growth factor. (D) HUVECs transfected with Dll4-Fl or vector alone were evaluated for cell proliferation (left panel) after 72 hours. Apoptosis in serum-deprived conditions was measured after 24 hours, using annexin-V FITC (right panel). Dll4-Fl induces fibronectin and artery specific genes. (E) HUVECs were transfected with expression vectors for Dll4-Fl or vector alone and sorted for transfected cells. Dll4 expression in Dll4-Fl transfected and sorted cells was assessed using MabDll4-FITC. cDNA was analyzed by RT-PCR for the expression of various genes that are differentially regulated in venous and arterial ECs. GAPDH and β-actin expression was examined to document equal amount of cDNA in each group. Two independent experiments produced similar results. EphB4, EphrinB2 and β-actin protein levels were evaluated by immunoblotting (bottom right panel). sDll4 induces vessel response in murine Matrigel assay. (F) Matrigel lacking growth factor or impregnated with VEGF or VEGF + sDll4-His were injected subcutaneously into Balb/C nu/nu mice. After 6 days, plugs were removed and processed in paraffin. Individual sections were stained with H&E, and representative photographs at 20× magnification from triplicate plugs in 2 independent experiments are shown. Matrigel plugs were stained for PECAM/CD31, or SMA. Photomicrographs were taken with an Olympus BX51 microscope with an Olympus UPlan FL, 0.17 20×/0.5 NA dry objective mounted with a Retiga 2000R camera (QImaging, Burnaby, BC) and processed with Image-Pro Plus 6.0 (Media Cybernetics). Quantitation of CD31, SMA positive cells was as in Figure 3. (G) Dll4-Fl induces fewer vessels with increased SMA. 293T cells were transfected with expression vectors for Dll4-Fl or vector alone and sorted cells were implanted in standard Matrigel in growth factor–deficient conditions in triplicates in 2 independent experiments. After 6 days, plugs were removed and processed in paraffin. Individual sections were stained with H&E, and representative photographs at 20× magnification from triplicate plugs in 2 independent experiments are shown. Matrigel plugs were stained for PECAM/CD31, or SMA. Photomicrographs were taken with an Olympus BX51 microscope with an Olympus UPlan FL, 0.17 20×/0.5 NA dry objective mounted with a Retiga 2000R camera (QImaging) and processed with Image-Pro Plus 6.0 (Media Cybernetics). Quantitation of CD31, SMA-positive cells was as in Figure 3.

Dll4-FL inhibits EC migration. (A) HUVECs were transfected with expression vectors for Dll4-Fl or vector alone and sorted for transfected cells. Confluent cultures of HUVECs were scraped with a plastic Pasteur pipette to produce a 3-mm–wide cell-free zone in the monolayer. The ability of the cells to migrate and close the wound was assessed over 18 hours. Dll4-Fl inhibited the migration of ECs even in the presence of VEGF, while exogenous sDll4 abolished this inhibition. (B) Endothelial cell migration into the cell free zone was quantitated using Bioquant Image Analysis (mean ± SEM from triplicate wells in 2 repetition experiments). *P < .05 when Dll4-Fl is compared with vector alone treated either with VEGF or sDll4-His. Dll4-Fl inhibits tubule formation in vitro. Photomicrographs were taken with a Nikon Plan Fluor, 0.17, 4×/0.12 NA objective and 10× eyepiece and processed with Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD). (C) HUVECs were transfected with expression vectors for Dll4-Fl or vector alone and sorted cells were cultured on standard Matrigel in growth factor–deficient conditions in triplicates in 2 independent experiments with either sDll4 or VEGF for 18 hours. Shown are representative pictures from triplicate wells repeated twice. Quantitative analysis for tube length and the number of junctions in various groups is presented. *P < .05 compared with no growth factor. (D) HUVECs transfected with Dll4-Fl or vector alone were evaluated for cell proliferation (left panel) after 72 hours. Apoptosis in serum-deprived conditions was measured after 24 hours, using annexin-V FITC (right panel). Dll4-Fl induces fibronectin and artery specific genes. (E) HUVECs were transfected with expression vectors for Dll4-Fl or vector alone and sorted for transfected cells. Dll4 expression in Dll4-Fl transfected and sorted cells was assessed using MabDll4-FITC. cDNA was analyzed by RT-PCR for the expression of various genes that are differentially regulated in venous and arterial ECs. GAPDH and β-actin expression was examined to document equal amount of cDNA in each group. Two independent experiments produced similar results. EphB4, EphrinB2 and β-actin protein levels were evaluated by immunoblotting (bottom right panel). sDll4 induces vessel response in murine Matrigel assay. (F) Matrigel lacking growth factor or impregnated with VEGF or VEGF + sDll4-His were injected subcutaneously into Balb/C nu/nu mice. After 6 days, plugs were removed and processed in paraffin. Individual sections were stained with H&E, and representative photographs at 20× magnification from triplicate plugs in 2 independent experiments are shown. Matrigel plugs were stained for PECAM/CD31, or SMA. Photomicrographs were taken with an Olympus BX51 microscope with an Olympus UPlan FL, 0.17 20×/0.5 NA dry objective mounted with a Retiga 2000R camera (QImaging, Burnaby, BC) and processed with Image-Pro Plus 6.0 (Media Cybernetics). Quantitation of CD31, SMA positive cells was as in Figure 3. (G) Dll4-Fl induces fewer vessels with increased SMA. 293T cells were transfected with expression vectors for Dll4-Fl or vector alone and sorted cells were implanted in standard Matrigel in growth factor–deficient conditions in triplicates in 2 independent experiments. After 6 days, plugs were removed and processed in paraffin. Individual sections were stained with H&E, and representative photographs at 20× magnification from triplicate plugs in 2 independent experiments are shown. Matrigel plugs were stained for PECAM/CD31, or SMA. Photomicrographs were taken with an Olympus BX51 microscope with an Olympus UPlan FL, 0.17 20×/0.5 NA dry objective mounted with a Retiga 2000R camera (QImaging) and processed with Image-Pro Plus 6.0 (Media Cybernetics). Quantitation of CD31, SMA-positive cells was as in Figure 3.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal