Influence of specific blocking of the delta-like ligand 4/notch signal transduction pathway on the biological behavior of human umbilical vein endothelial cells.
The influence of specific blocking of the Delta-like ligand 4 (DLL4)/Notch signal transduction pathway on the biological behavior of human umbilical vein endothelial cells (HUVECs) has been studied. Recombinant adeno-associated virus (rAAV) vectors expressing an active small interfering RNA (siRNA) (vector 6) targeting the DLL4 (rAAV-DLL4-short hairpin RNA [shRNA]) was used to infect HUVECs. The same cell line infected with empty plasmid (rAAV-EGFP) was used as a control. Stable transfection and expression of DLL4-mRNA in HUVECs were determined by semiquantitative RT-polymerase chain reaction (PCR). Protein expression of DLL4 was examined by western blotting. The distribution of cells in cell cycle was assessed by flow cytometry and cell growth was analyzed by MTT assay. HUVECs were seeded on type I collagen and cultured in a three-dimensional culture system to allow for tubule-like structure (TLS) formation. Compared with negative controls, semiquantitative RT-PCR and western blot analysis showed that the expression of DLL4 mRNA and protein was downregulated in stably transfected cells (p = 0.024, p = 0.033). HUVEC growth and proliferation were stimulated following infection with rAAV vectors containing active siRNA against DLL4, whereas infection with empty plasmid had no specific effect. The proliferation index of rAAV-DLL4-shRNA-infected HUVECs was 39.90% +/- 2.19% compared with 25.63% +/- 4.54% (p = 0.036) for control-treated cells. TLS formation was significantly induced in cells expressing the rAAV vector; the average length of TLS was greater than the control group (p = 0.028). Altogether, the data suggest that inhibiting the DLL4/Notch signal transduction pathway stimulated proliferation of HUVECs, thereby facilitating angiogenesis.
Lv
Wei
W
Department of General Surgery, The Second Artillery General Hospital, Beijing, China.
Chen
Lin
L
Zhou
Ding-Hua
DH
Wei
Bo
B
D048868:Adaptor Proteins, Signal Transducing; D015153:Blotting, Western; D002135:Calcium-Binding Proteins; D002453:Cell Cycle; D049109:Cell Proliferation; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000166:cytology*; D005434:Flow Cytometry; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D018919:Neovascularization, Physiologic; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics*; D014471:Umbilical Veins / Q000166:cytology
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; C424126:DLL4 protein, human; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051880:Receptors, Notch