[Influence of Delta-like ligand 4/Notch signal transduction pathway upon the biological behavior of human umbilical vein endothelial cells]

Abstract

Objective: To study the influence of inhibiting Delta-like ligand 4 (Dll4)/Notch signal transduction pathway upon the biological behavior of human umbilical vein endothelial cells (HUVEC).

Methods: Used rAAV vectors expressing an active small interfering RNA (siRNA) (vector 6) targeting the Dll4 (rAAV-Dll4-shRNA) to infect HUVEC. And an empty plasmid (rAAV-EGFP) was infected into the same cell line as control group. The stable transfection and expression of Dll4 mRNA in HUVEC were determined by semi-quantitative RT-PCR. The protein expression of Dll4 was examined by Western blotting. Distribution of cell cycle was assessed by flow cytometry. The cell growth was analyzed by MTT assay. HUVEC were separated by type I collagen and cultured in a three-dimensional culture system for tubule like structure (TLS) formation.

Results: Compared with the negative control cells, semi-quantitative RT-PCR and Western blotting showed the expression of Dll4 mRNA (0.636 +/- 0.082, 0.972 +/- 0.022 vs 0.948 +/- 0.046) and protein (0.632 +/- 0.052, 2.016 +/- 0.048 vs 1.946 +/- 0.066) were down-regulated in the stable cell (P = 0.024, 0.033). The rAAV vectors expressing an active small interfering RNA (siRNA) targeting the Dll4 effectively stimulated HUVEC cell growth and proliferation while empty plasmid had no such specific effect. The proliferation index of experimental group was (39.9 +/- 2.2)% versus untreated group (25.7 +/- 4.5)% (P = 0.036). TLS formation was significantly induced by rAAV vector. And the average length of TLS were more than those of control group (12.5 +/- 0.5, 8.7 +/- 7.7, 8.5 +/- 3.0, P = 0.028).

Conclusion: The inhibiting Dll4/Notch signal transduction pathway stimulates the proliferation of HUVEC and facilitates the angiogenesis. Interference with Dll4/Notch signaling may be particularly desirable in tumors with highly induced Dll4/Notch pathway.