Effect of VEGF-C production mediated by SiO₂ stimulated U937 cells on tubular structure formation of human lymphatic endothelial cells

Background Our preliminary animal experiment results show that silica (SiO₂) induces significant lymphangiogenesis in rat lung tissues in early stage, which may be of great significance in removing lung dust and alleviating the pathogenesis of silicosis. However, the specific molecular mechanism of lymphangiogenesis has not been fully understood.

Objective This experiment explores the effect of SiO₂ stimulated human mononuclear macrophages U937 producing vascular endothelial growth factor C (VEGF-C) on the tubular structure formation of human lymphatic endothelial cells (HLECs).

Methods U937 cells were stimulated with different concentrations of SiO₂ (0, 25, 50, 100, and 200 mg·L^-1) for different time (12, 24, and 48 h). Western blotting and ELISA were used to detect the VEGF-C protein level in U937 cells and in cell culture supernatant respectively. The combination of SiO₂ stimulation concentration and time generating the highest VEGF-C protein expression level was selected for further experiment, and the cell culture supernatant was collected as conditioned medium (CM). HLECs were divided into four groups. The CM treatment group was treated with CM stimulated by SiO₂, the VEGF-C treatment group with a medium containing 5000ng·L^-1 human VEGF-C recombinant protein, the CM control group with cell culture supernatant without SiO₂ stimulation, and the negative control group without any administration. The cell proliferation of HLECs was detected by MTS method after the cells were treated by 10%, 50%, and 100% CM for 24, 48, and 72 h; the cell migration and tubular structure formation were detected by scratch test and matrigel tube formation test after the cells were treated by 100% for 12 h and 6 h respectively.

Results The SiO₂ stimulation at 50 mg·L^-1 for 24 h produced the highest VEGF-C protein level in both U937 cells and cell culture supernatant(1.12±0.08) and (5 464.00±231.21) ng·L^-1 respectively compared with the 0 mg·L^-1 group. With time extension (24, 48, and 72 h), the CM concentration was higher, and the proliferation of HLECs was enhanced. The 100% CM treatment group at 72 h had the highest cell proliferation (2.00±0.13), the VEGF-C treatment group at 72 h also showed increased proliferation (1.67±0.10), and the differences were significant compared with the control group (P < 0.05). The migration area ratios of HLECs in the CM treatment group and the VEGF-C treatment group(53.64±4.74)% and (56.82±5.72)% respectively were increased compared with the control group (P < 0.05). The number of tubes (7.20±1.30, 8.20±1.64) and the number of tube branches (32.60±5.13, 38.20±8.70) of HLECs were increased in the CM treatment group and the VEGF-C treatment group compared with the control group (P < 0.001).

Conclusion SiO₂ may promote the proliferation, migration, and tubular structure formation of HLECs by stimulating human mononuclear macrophages U937 to produce VEGF-C.