Slit2 suppresses endothelial cell proliferation and migration by inhibiting the VEGF-Notch signaling pathway

Abstract

Slit homolog 2 (Slit2) is distributed in various tissues and participates in numerous cellular processes; however, the role of Slit2 in the regulation of angiogenesis remains controversial, since it has previously been reported to exert proangiogenic and antiangiogenic activities. The present study aimed to investigate the effects of Slit2 on vascular endothelial cell proliferation and migration in vitro, and to reveal the possible underlying signaling pathway. Aortic endothelial cells were isolated from Sprague Dawley rats and cultured. Cell proliferation assay, cell migration assay, immunocytochemistry and small interfering RNA transfection were subsequently performed. The results demonstrated that exogenous Slit2 administration markedly suppressed TNF‑α‑induced endothelial cell proliferation and migration in vitro. In addition, TNF‑α application upregulated the protein expression levels of vascular endothelial growth factor (VEGF) and Notch in RAECs, whereas Slit2 administration downregulated VEGF and Notch expression in RAECs cultured in TNF‑α conditioned medium. Further studies indicated that knockdown of VEGF suppressed the effects of TNF‑α on the induction of RAEC proliferation and migration. VEGF knockdown‑induced inhibition of RAEC proliferation and migration in TNF‑α conditioned medium was also achieved without Slit2 administration. Furthermore, VEGF knockdown markedly decreased Notch1 and Notch2 expression. These results indicated that Slit2 suppresses TNF‑α‑induced vascular endothelial cell proliferation and migration in vitro by inhibiting the VEGF‑Notch signaling pathway. Therefore, Slit2 may inhibit the proliferation and migration of endothelial cells during vascular development.

Figure 1.

TNF-α induced cell proliferation of RAECs. (A) Time-course and dose-response of RAEC proliferation following treatment with TNF-α. (B) Number of viable cells at 48 h following treatment with various concentrations of TNF-α. RAECs were seeded in a 96-well plate and were treated with various doses of TNF-α for 48 h; the number of viable cells was determined at the end of the experiment. Data are presented as the mean ± standard deviation of 6–10 individual samples of independent triplicate experiments. *P<0.05 vs. control group. RAECs, rat aortic endothelial cells; TNF-α, tumor necrosis factor-α.

Figure 2.

TNF-α enhanced migratory ability of RAECs. (A) Migration was detected using a Transwell assay (magnification, ×100). (B) Compared with the control group, the migratory ability of RAECs was significantly increased in the TNF-α conditioned medium group. A concentration of 10 ng/ml TNF-α was used for the migration analysis. The control group was incubated in endothelial cell medium. Data are presented as the mean ± standard deviation of 6 individual samples of independent triplicate experiments. *P<0.05 vs. control group. RAECs, rat aortic endothelial cells; TNF-α, tumor necrosis factor-α.

Figure 3.

TNF-α increased VEGF, Notch1 and Notch2 expression in RAECs. (A) Protein and mRNA expression levels were determined by western blotting and polymerase chain reaction, respectively. (B) VEGF, Notch1 and Notch2 protein expression was significantly increased in RAECs following treatment with 10 ng/ml TNF-α for 48 h. (C) Compared with the control group, VEGF, Notch1 and Notch2 mRNA expression levels were also significantly increased in RAECs in the TNF-α conditioned medium group. GAPDH was used as an endogenous control and the control group was used as a calibrator sample. *P<0.05 vs. control group; n=6/group. The experiment was performed in triplicate. RAECs, rat aortic endothelial cells; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor.

Figure 4.

Slit2 decreased TNF-α-induced cell proliferation and migration in RAECs. (A and B) RAEC migration was detected by Transwell analysis (magnification, ×100). RAEC migratory ability was improved by 10 ng/ml TNF-α; however, Slit2 attenuated the increase in migratory ability. As the added concentration of Slit2 increased, the TNF-α-induced migratory ability of RAECs was markedly inhibited. (C) RAEC proliferation was increased by 10 ng/ml TNF-α; however, Slit2 ameliorated proliferation in a dose-dependent manner. *P<0.05 vs. control group; ▲P<0.05 vs. TNF-α group; ^◊P<0.05 vs. 25 ng/ml Slit2 group; ▼P<0.05 vs. 50 ng/ml Slit2 group. RAECs, rat aortic endothelial cells; TNF-α, tumor necrosis factor-α; Slit2, Slit homolog 2.

Figure 5.

Slit2 attenuated TNF-α-induced increases in VEGF and Notch expression. (A) VEGF, Notch1 and Notch2 protein expression, as determined by western blotting. (B) VEGF, Notch1 and Notch2 mRNA expression, as determined by polymerase chain reaction. Rat aortic endothelial cells were treated with 10 ng/ml TNF- for 48 h, and the protein and mRNA expression levels of VEGF, Notch1 and Notch2 were detected. TNF-α induced VEGF, Notch1 and Notch2 overexpression was inhibited by Slit2 administration. *P<0.05 vs. control group; ▲P<0.05 vs. TNF-α group; ♦P<0.05 vs. 25 ng/ml Slit2 group; ▼P<0.05 vs. 50 ng/ml Slit2 group. TNF-α, tumor necrosis factor-α; Slit2, Slit homolog 2.

Figure 6.

Inhibitory effects of Slit2 on TNF-α-induced cell proliferation and migration were mediated via the VEGF-Notch pathway. (A) VEGF levels. RAECs were treated with 10 ng/ml TNF-α or 100 ng/ml Slit2, in the presence or absence of VEGF siRNA (200 nM). TNF-α induced an increase in VEGF levels; however, Slit2 and VEGF siRNA reduced the expression levels of VEGF [*P<0.05 vs. TNF-α(+)/Slit2(−)/VEGF(−)/NS siRNA (−)-transfected cells]. (B) Notch1 and Notch2 protein expression levels were markedly decreased following VEGF siRNA transfection. (C) RAEC proliferation. Slit2 and VEGF siRNA transfection inhibited TNF-α-induced cell proliferation [♦P<0.05 vs. TNF-α(+)/Slit2(−)/VEGF(−)/NS siRNA(−)-transfected cells]. (D) RAEC migration. Slit2 and VEGF siRNA transfection inhibited TNF-α-induced cell migration [♦P<0.05 vs. TNF-α(+)/Slit2(−)/VEGF(−)/NS siRNA(−)-transfected cells]. RAECs, rat aortic endothelial cells; TNF-α, tumor necrosis factor-α; Slit2, Slit homolog 2; VEGF, vascular endothelial growth factor; NS, non-specific.