title: Toxic effects of recombinant tissue plasminogen activator on cultured human corneal endothelial cells.
abstract: OBJECTIVE
  Human corneal endothelial cells (HCECs) are nonmitotic cells. Any intracameral application of a drug requires evaluation of the potential apoptotic and toxic effects. Intracameral recombinant tissue plasminogen activator (rtPA) is successfully used for the treatment of severe and prolonged postoperative fibrin reaction. This study was undertaken to investigate the toxicity of rtPA on cultured HCECs to determine its safety for clinical use.
  
  
  METHODS
  Cell cultures of HCECs were harvested from human donor eyes and exposed to various concentrations of rtPA (10-200 microg/mL). For cytotoxicity testing, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and the live/dead viability/cytotoxicity assay were performed. Annexin V binding combined with propidium iodide (PI) costaining was used for the distinction of viable, early, and late apoptotic cells. Odds ratios (ORs) and confidence intervals (CIs) were calculated for 50 microg/mL, 100 microg/mL, and 200 microg/mL. Cell morphology was studied after 24 hours of exposure to rtPA to identify cellular damage. Immunolocalization of zonula occludens 1 (ZO1) was performed to analyze intercellular barrier disturbance in the presence of rtPA.
  
  
  RESULTS
  Reduction of mitochondrial dehydrogenase activity after rtPA exposure was dose dependent and suggested comparable toxicity with the data obtained from the live/dead assay. The mean number of Annexin V-FITC and PI-positive cells was not significantly increased at concentrations of 50 microg/mL and 100 microg/mL. At 200 microg/mL, however, the ORs were 5.082 +/- 0.213 (95% CI, 3.962-6.203; P < 0.001) for apoptosis and 6.154 +/- 0.196 (95% CI, 5.123-7.181; P < 0.001) for necrosis. In addition, increasing concentrations of rtPA resulted in a fading immunopositive staining for ZO1.
  
  
  CONCLUSIONS
  These data suggest a dose-dependent toxic effect of rtPA on HCECs in vitro. Although intracameral rtPA concentrations up to 100 mug/mL seem to be clinically safe, the use of concentrations higher than 125 microg/mL might induce irreversible cell death and should be restricted to selected cases.
authors.affiliation: Department of Ophthalmology, Eberhard-Karls University, Tuebingen, Germany.
authors.forename: Efdal
authors.initials: E
authors.lastname: Yoeruek
authors.forename: Martin S
authors.initials: MS
authors.lastname: Spitzer
authors.forename: Olcay
authors.initials: O
authors.lastname: Tatar
authors.forename: Tilo
authors.initials: T
authors.lastname: Biedermann
authors.forename: Salvatore
authors.initials: S
authors.lastname: Grisanti
authors.forename: Matthias
authors.initials: M
authors.lastname: Lüke
authors.forename: Karl U
authors.initials: KU
authors.lastname: Bartz-Schmidt
authors.forename: Peter
authors.initials: P
authors.lastname: Szurman
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D002470:Cell Survival; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004728:Endothelium, Corneal / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques; D008565:Membrane Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000633:toxicity*; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000633:toxicity*; D062826:Zonula Occludens-1 Protein
chemical_list: D017304:Annexin A5; D008565:Membrane Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C569042:TJP1 protein, human; D013778:Tetrazolium Salts; D013844:Thiazoles; D062826:Zonula Occludens-1 Protein; D010959:Tissue Plasminogen Activator; C022616:thiazolyl blue