title: Inhibitory effect of rapamycin on corneal neovascularization in vitro and in vivo.
abstract: 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.
authors.affiliation: Department of Ophthalmology, College of Medicine, Chung-Ang University, Seoul 140-757, Korea.
authors.forename: Young Sam
authors.initials: YS
authors.lastname: Kwon
authors.forename: Hyun Sook
authors.initials: HS
authors.lastname: Hong
authors.forename: Jae Chan
authors.initials: JC
authors.lastname: Kim
authors.forename: Jun Seop
authors.initials: JS
authors.lastname: Shin
authors.forename: Youngsook
authors.initials: Y
authors.lastname: Son
mesh_terms: 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
chemical_list: 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