Suppression of Human Tenon Fibroblast Cell Proliferation by Lentivirus-Mediated VEGF Small Hairpin RNA

Abstract

Purpose. The functions of vascular endothelial growth factor (VEGF) in scar formation after trabeculectomy were investigated in a human Tenon fibroblast cell line from glaucoma patients using lentivirus-mediated VEGF shRNA. Methods. Human Tenon fibroblast (HTF) cells were isolated from scar tissue of glaucoma patients during secondary surgery. Lentivirus-VEGF-shRNA was constructed and transfected into HTF cells. Subsequently, VEGF mRNA and protein expression were analyzed using quantitative RT-PCR and western blotting, respectively, and the effects of VEGF knockdown were analyzed. The inhibition of HTF proliferation was monitored according to total cell numbers using ScanArray. Results. Both mRNA and protein levels of VEGF were reduced by lentivirus-mediated VEGF-shRNA, and proliferation of HTF cells was inhibited. Conclusions. Primary cultures of human Tenon fibroblast (HTF) were established, and proliferation was decreased following inhibition of VEGF. VEGF may be a suitable therapeutic target for reducing scar tissue formation in glaucoma patients after filtration surgery.

Figure 1

RNA interference after transfection with VEGF-shRNA. ^∗∗ p < 0.1; ^∗∗∗ p < 0.01.

Figure 2

Western blot analysis of the effects of RNA interference on VEGF expression in HTF cells. (a) Relative expression of VEGF in cells treated with shRNA-2 and shRNA-3; (b) protein expression levels in treated groups and naïve HTF controls. ^∗∗∗ p < 0.01.

Figure 3

Cell growth curves. Total cell numbers were counted at 4, 7, 11, and 14 days after addition of DOX to cultures. Curves represent total cell numbers of naïve HTF cells, vehicle transfected HTF cells, VEGF-shRNA-2 transfected HTF cells, and VEGF-shRNA-3 transfected HTF cells. ⁎ means p value < 0.5 compared to vehicle control. ⁎⁎ means p value < 0.1 compared to vehicle control.