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Z.-J. Du, M. Kamei, M. Suzuki, K. Nishida, H. Sakaguchi, P. Xie, Y. Tano; Activated Protein C Inhibits Tissue Plasminogen Activator-Induced Retinal Toxicity. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4885.
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Tissue plasminogen activator (tPA) has been applied for several ocular disorders, although excessive doses of intravitreal tPA cause retinal toxicity. Previous works demonstrated that tPA toxicity to brain tissues increases under ischemic conditions and that activated protein C (APC) inhibits tPA toxicity. The purpose of this study was to analyze tPA toxicity in the ischemic retina and to evaluate the protective effect of APC against tPA toxicity in the retina.
Retinal pigment epithelium cells (ARPE-19) were cultured, and placed either under a normoxic condition or in a hypoxic chamber to induce an ischemic condition. The cultures were then treated with 2.5, 5, 10, 20, or 40 µg/ml of tPA for 24 hours, followed by MTT assay and TUNEL staining to determine the number of viable cells and apoptotic cells, respectively. Activity of caspase 3, 8, and 9 was also analyzed. To examine the potential inhibitory effect of APC against tPA-induced cyototoxicity, the cultures were treated with APC (ranging from 3 to 240 µg/ml) alone or with a mixture of tPA with APC. To detect the in vivo effects of tPA and APC, various concentrations of tPA, APC, and the mixture were intravitreally injected in a rat central retinal vein occlusion (CRVO) model, followed by TUNEL staining 24 hours after the injection.
The number of dead cells increased in a dose-dependent manner under a normoxic condition when treated with 20 µg or greater tPA and significantly increased under a hypoxic condition (P=0.002). Lower concentrations of APC (ranging from 3 to 60 µg/ml) showed a cell protective effect against hypoxia, while higher concentrations of APC (120 µg/ml or greater) demonstrated cytotoxicity. TPA-induced cytotoxicity was significantly suppressed by 6 µg/ml of APC both in vitro and in vivo. Caspase 3, 8, and 9 were activated when cells were subjected to hypoxia or treated with tPA, and this activation was significantly inhibited by APC (P<0.001).
APC can reduce the cytotoxicity induced by tPA both in vitro and in vivo via blocking the activation of caspase 3, 8, and 9. APC might be a promising candidate not only to suppress tPA cytotoxicity but also to protect the retina from ischemic conditions.
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