Toxic effects of recombinant tissue plasminogen activator on cultured human corneal endothelial cells

Purpose. Human corneal endothelial cells (HCECs) are nonmitotic cells. Any intracameral application of a drug requires evaluation of the potential apoptotic and toxic effects. Intra- cameral 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 μg/mL). For cytotoxicity testing, the 3-(4,5-dimethyl-thiazol-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 μg/mL, 100 μg/mL, and 200 μg/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 μg/mL and 100 μg/mL. At 200 μg/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 μg/mL seem to be clinically safe, the use of concentrations higher than 125 μg/mL might induce irreversible cell death and should be restricted to selected cases.