Inhibitory effect of rapamycin on corneal neovascularization in vitro and in vivo.
OBJECTIVE
To examine the effect of rapamycin on the proliferation and the migration of human umbilical vein endothelial cells (HUVECs) and on the corneal neovascularization in the corneal alkaline burn murine model.


METHODS
HUVEC proliferation, migration, and apoptosis were examined after treatment with rapamycin. The effect of rapamycin on the mRNA expression of FK506 binding protein (FKBP)-12 and mammalian target of rapamycin (mTOR) was also evaluated in vitro. Corneal neovascularization was induced in vivo by an alkaline burn of the cornea with 1 N NaOH on BALB/c mice. Rapamycin was given intraperitoneally at 2 mg/kg body weight once a day for 12 days after the corneal alkaline burn. Growth factors and cytokines related with neovascularization and inflammation were evaluated in the corneal tissue and the peripheral blood by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The corneal neovascularization was evaluated by a slit lamp biomicroscopy.


RESULTS
Rapamycin at the concentration of 1000 ng/mL for >48 hours' exposure significantly inhibited the growth of HUVECs. The double chamber assay showed that rapamycin dramatically inhibited the migration of HUVECs at concentrations of 10 and 100 ng/mL and that these concentrations did not affect endothelial cell growth. When TUNEL assays were performed, the number of apoptotic cells increased 1.9-, 2.1-, and 2.6-fold compared with the control at 10, 100, and 1000 ng/mL, respectively, of rapamycin at 48 hours of exposure. RT-PCR showed that the expression of mTOR was suppressed in the HUVECs after rapamycin treatment; however, FKBP-12 expression was not affected. Among the angiogenic factors, gene expression of substance P and hypoxia inducible factor (HIF)-1 alpha was inhibited by rapamycin earlier (1-3 days), with vascular endothelial growth factor (VEGFR)-1 gene expression being suppressed for the first 7 days in the corneal tissue. The protein level of substance P and vascular endothelial growth factor (VEGF) was significantly decreased--more in mice treated with rapamycin than the control mice--as shown by ELISA assay of peripheral blood. Furthermore, rapamycin significantly inhibited corneal neovascularization in the alkaline-burned cornea.


CONCLUSIONS
Rapamycin strongly inhibited HUVEC migration at doses that did not cause cytotoxicity and apoptosis in this in vitro model. Rapamycin also suppressed corneal neovascularization, possibly by inhibiting proinflammatory cytokines, as shown by the in vivo study. Therefore, rapamycin may be useful as an angiogenic regulator in the treatment of corneal diseases that manifest with neovascularization.
Kwon
Young Sam
YS
Department of Ophthalmology, College of Medicine, Chung-Ang University, Seoul 140-757, Korea.
Hong
Hyun Sook
HS
Kim
Jae Chan
JC
Shin
Jun Seop
JS
Son
Youngsook
Y
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D016510:Corneal Neovascularization / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control*; D016207:Cytokines / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006133:Growth Substances / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D020287:In Situ Nick-End Labeling; D007274:Injections, Intraperitoneal; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020123:Sirolimus / Q000494:pharmacology*; D012972:Sodium Hydroxide; D058570:TOR Serine-Threonine Kinases; D014471:Umbilical Veins / Q000166:cytology
D002352:Carrier Proteins; D016207:Cytokines; D006133:Growth Substances; D007166:Immunosuppressive Agents; D012333:RNA, Messenger; D012972:Sodium Hydroxide; D011494:Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C546842:MTOR protein, human; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus