Rapamycin prevents endothelial cell migration by inhibiting the endothelial-to-mesenchymal transition and matrix metalloproteinase-2 and -9: an in vitro study - PubMed (original) (raw)

Rapamycin prevents endothelial cell migration by inhibiting the endothelial-to-mesenchymal transition and matrix metalloproteinase-2 and -9: an in vitro study

Hua Gao et al. Mol Vis. 2011.

Abstract

Purpose: To evaluate the influence of rapamycin on endothelial-mesenchymal transition and matrix metalloproteinase (MMP) secretion by human umbilical vein endothelial cell line EA.hy926 and explore rapamycin's angiogenesis inhibition mechanism.

Methods: EA.hy926 cells were cultivated in vitro. After the cells attained complete confluency, an artificial scratch was made through the monolayer with a sterile plastic 100 μl micropipette tip. Cell morphology changes were observed. The expression of vascular endothelial (VE)-cadherin, vimentin, and Twist protein were examined by immunofluorescence. After scratching, the cells were treated with 10, 100, and 1,000 ng/ml rapamycin for durations of 24, 48, and 72 h. Cell proliferation was then assessed using methyl thiazolyl tetrazolium assay. Cell migration ability was examined, and the expression of VE-cadherin, vimentin, and the Twist transcription factor in mRNA levels was evaluated with reverse transcriptase PCR. The expression of gelatinases (MMP-2 and MMP-9) was examined using gelatin zymography.

Results: After scratching, the endothelial cells were able to migrate via an endothelial-to-mesenchymal transition, which was related to Twist expression. Finally, mesenchymal cells transitioned into endothelial cells and reached cell confluency again. The growth of EA.hy926 cells was not affected by rapamycin concentrations of 10 ng/ml or 100 ng/ml during treatment periods of 1, 2, and 3 days; however, cell growth was inhibited by 1,000 ng/ml rapamycin with a three-day treatment period. Rapamycin successfully inhibited cell migration at concentrations of 10 ng/ml, 100 ng/ml, and 1,000 ng/ml for a treatment period of up to 8 h. Different concentrations of rapamycin induced the expression of VE-cadherin, inhibited vimentin and Twist expression in the endothelial cells, and inhibited endothelial cell secretion of MMP-2 and MMP-9.

Conclusions: Rapamycin inhibited cell migration and extracellular matrix degradation by inhibiting endothelial-to-mesenchymal transition and the endothelial cell secretion of MMP-2 and MMP-9; these may be possible mechanisms for the inhibition of angiogenesis by rapamycin.

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Figures

Figure 1

Cell morphology. A: In the no-scratch group, after cell confluency, EA.hy926 cells formed a cobblestone-like monolayer. B: In the scratch group, the endothelial cells that migrated into the scratch wound showed a fibroblast-like morphology with visible pseudopods (contrast phase microscope 100×).

Figure 2

Immunofluorescence images. A: In the no-scratch group, after cell confluency, vascular endothelial (VE)-cadherin yielded continuous, linear staining around the periphery of EA.hy926 cells (confocal microscopy 200×). B: In the no-scratch group, after cell confluency, there was no expression of vimentin protein (confocal microscopy 100×). C: In the no-scratch group, after cell confluency, there was no expression of the Twist protein (confocal microscopy 100×). D: In the scratch group, the VE-cadherin expression of the endothelial cells that migrated into the scratch wound was decreased at endothelial cell-cell junctions and was mainly localized to the cytoplasm; VE-cadherin yielded continuous, linear staining around the periphery of EA.hy926 cells on the outside of the scratch wound (confocal microscopy 100×). E: In the scratch group, the vimentin expression of endothelial cells that migrated into the scratch wound (confocal microscopy 100×). F: In the scratch group, the Twist expression of endothelial cells that migrated into the scratch wound (confocal microscopy 100×).

Figure 3

The effect of rapamycin on EA.hy926 cell growth. EA.hy926 cells were treated with 0 to 1,000 ng/ml rapamycin for 24, 48, or 72 h. Cell growth was determined by methyl thiazolyl tetrazolium assay (MTT). 0 ng/ml of rapamycin represents the control group. Bars represent standard deviation (SD; n=6 wells per measurement). Similar results were obtained in three independent experiments, and the data were shown as mean±SD. The asterisk indicates p<0.05, compared with the control group with independent samples _t_-test.

Figure 4

The effect of rapamycin on the migration of EA.hy926 cells. EA.hy926 cells were treated with 0 to 1,000 ng/ml rapamycin for 8 h. Cell migration ability was determined by the scratch wound assay method. 0 ng/ml of rapamycin represents the control group. Bars represent SD (n=6 photographs per measurement). Similar results were obtained in three independent experiments, and the data were shown as mean±SD. The asterisk indicates p<0.05, compared with the control group with independent samples _t_-test.

Figure 5

Reverse transcriptase–PCR gel electrophoresis images of vascular endothelial–cadherin/vimentin/Twist/glyceraldehyde 3-phosphate dehydrogenase mRNA expression in EA.hy926 cells. The effect of rapamycin on the gene expression of vascular endothelial (VE)-cadherin, vimentin, and Twist. (a, scratch group; b1, 10 ng/ml rapamycin group; b2, 100 ng/ml rapamycin group; b3, 1,000 ng/ml rapamycin group; c, no-scratch group).

Figure 6

Relative expression ratio of the target gene versus the housekeeping gene (glyceraldehyde 3-phosphate dehydrogenase). The ratio for the vertical axis is relative expression ratio of the target gene versus the house-keeping gene (glyceraldehyde 3-phosphate dehydrogenase [_GAPDH_]). Three independent experiments were conducted. Bars represent standard deviation (SD), and the data were shown as mean±SD. The asterisk indicates p<0.05 in comparison between each rapamycin group and the scratch group with independent samples _t_-test. Pound sign (#) indicates p<0.05 in comparison between the scratch group and the no-scratch group with independent samples _t_-test. a, scratch group; b1, 10 ng/ml rapamycin group; b2, 100 ng/ml rapamycin group; b3, 1,000 ng/ml rapamycin group; c, no-scratch group.

Figure 7

Gelatin zymography gel electrophoresis images of matrix metalloproteins 2 and 9 (a, scratch group; b1, 10 ng/ml rapamycin group; b2, 100 ng/ml rapamycin group; b3, 1,000 ng/ml rapamycin group; c, no-scratch group).

Figure 8

The result of densitometry values of MMP 2 and 9. The effect of rapamycin on the expression of matrix metalloprotein (MMP)-2 and −9 was determined by Gelatin zymography. Three independent experiments were conducted. Bars represent standard deviation (SD), and the data were shown as mean±SD. The asterisk (*) indicates p<0.05 in comparison between each rapamycin group and the scratch group with independent samples _t_-test. a, scratch group; b1, 10 ng/ml rapamycin group; b2, 100 ng/ml rapamycin group; b3, 1000 ng/ml rapamycin group; c, no-scratch group.

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Rapamycin prevents endothelial cell migration by inhibiting the endothelial-to-mesenchymal transition and matrix metalloproteinase-2 and -9: an in vitro study - PubMed (original) (raw)