Inhibition of adenovirus-mediated recombinant Tum5 gene overexpression on human umbilical vein endothelial cells in physiological status

Background

Tumstatin is the most active endogenous angiogenesis inhibitor, which has a marked inhibitory effect on pathological neovascularization, and Tum5 is an angiogenesis inhibitors fragment of full-length tumstatin.

Objective

This study was to investigate the effects of adenovirus-mediated overexpression of recombinant Tum5 gene on the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) in physiological status.

Methods

The empty adenoviral vector expressing green fluorescent protein (rAd-GFP) and the viral vector expressing recombinant Tum5 gene were constructed.The HUVECs cultured in RPMI1640 medium were divided into normal control group, empty vector group (rAd-GFP group) and Tum5 gene infection group (rAd-GFP-Tum5 group). The rAd-GFP and rAd-GFP-Tum5 adenoviral particles at the density of 1×10¹⁰/ml were added into the medium to infect the cells for 48 hours.The proliferation of the cells was assayed at 24, 48 and 72 hours by cell counting kit-8 (CCK-8) to evaluate the proliferative rate; the migration number of the cells was detected at 48 hours after infection by Transwell chamber; the tube formation number of the cells were detected by Matrigel method.The concentration of vascular endothelial growth factor (VEGF) in cell supernatants was assayed by ELISA at 24, 48, and 72 hours following adenoviral infection.

Results

The cultured cells showed green fluorescence in the rAd-GFP group and rAd-GFP-Tum5 group under the inverted fluorescence microscope, and the infection efficiency of rAd-GFP and rAd-GFP-Tum5 was 55.13% and 50.31%, respectively.No significant difference was found in cell proliferative rate among normal control group, rAd-GFP group and rAd-GFP-Tum5 group both at 24 and 48 hours after infection (both at P>0.05), and the cell proliferative rate was significantly lower in the rAd-GFP-Tum5 group than that in the normal control group and rAd-GFP group at 72 hours after infection (both at P<0.01). The migration number of the cells at 48 hours after infection was 2 260.25±930.44, 2 370.00±441.06 and 723.75±363.80 in the normal control group, rAd-GFP group and rAd-GFP-Tum5 group, showing a significant difference among the groups (F=8.524, P=0.008), and the migrated cells were evidently decreased in the rAd-GFP-Tum5 group compared with the rAd-GFP group and the normal control group (both at P< 0.01 ). The tube number at 48 hours after infection was 95.67±5.86, 88.00±4.58 and 20.67±3.51 in the normal control group, rAd-GFP group and rAd-GFP-Tum5 group, showing a significant difference among the groups (F=226.498, P<0.01), and the tube number in the rAd-GFP-Tum5 group was significantly reduced in comparison with the normal control group and rAd-GFP group (both at P<0.01). The considerably differences in VEGF concentration in the cell supernatants were found in different groups and various time points (F_group=73.260, P<0.01; F_time=73.477, P<0.01), and VEGF concentration in the cell supernatants was significantly decreased in the rAd-GFP-Tum5 group compared with the rAd-GFP group at both 48 hours and 72 hours (both at P<0.01).

Conclusions

The overexpression of the recombinant Tum5 can inhibit the proliferation, migration and tube formation of the HUVECs in physiological status, which may be associated with Tum-5-mediated down-regulation of VEGF protein in the cell supernatant.