Rapamycin prevents endothelial cell migration by inhibiting the endothelial-to-mesenchymal transition and matrix metalloproteinase-2 and -9: an in vitro study.
OBJECTIVE
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.
State Key Lab Cultivation Base, Shandong Provincial Key Lab of Ophthalmology, Shandong Eye Institute, Qingdao, China.
Hua
H
Gao
Jingjing
J
Zhang
Ting
T
Liu
Weiyun
W
Shi
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D054338:Cell Transdifferentiation / Q000187:drug effects*; D002478:Cells, Cultured; D015870:Gene Expression / Q000187:drug effects*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020123:Sirolimus / Q000494:pharmacology*; D051879:Twist-Related Protein 1 / Q000235:genetics / Q000378:metabolism; D014746:Vimentin / Q000235:genetics / Q000378:metabolism; D000099152:Cadherin 5
D020533:Angiogenesis Inhibitors; D015703:Antigens, CD; D015820:Cadherins; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D009687:Nuclear Proteins; D012333:RNA, Messenger; D020123:Sirolimus; D051879:Twist-Related Protein 1; D014746:Vimentin; D000099152:Cadherin 5; C103173:TWIST1 protein, human