Urokinase Gene Expression in Cultured Human Trabecular Meshwork Cells and Gene Transfer to Porcine Cells

Abstract

Abstract: : Purpose: We investigated the gene expression of urokinase (uPA) in cultured human trabecular meshwork (TM) cells and determined whether uPA gene transfer into porcine TM cells is possible. Methods: Human TM cells were obtained from POAG patients undergoing a trabeculectomy and porcine TM cells were freshly isolated from eye specimens. Both types of cells were cultured in 5% fetal bovine serum added to F12/DME medium and used at the second or third passage. Total RNA was extracted from human TM cells and reverse transcribed into cDNA. A reverse–transcribed polymerase chain reaction method was performed to detect the gene expression of uPA using specific primers for human cDNA sequences. The complete sequenced cDNA of human uPA was then sub–cloned into an expression vector and transfected into the cultured porcine TM cells using lipofectamine. As a control, the expression vector alone was also transfected into cultured cells. Expression of the transferred human uPA was examined using an enzyme linked immunosorbent assay (ELISA). Results: uPA gene expression was observed in the cultured human TM cells, whereas the porcine TM cells exhibited no band in the PCR assays using primers for human uPA. However, ELISA revealed expression of the transferred human uPA (3.5 ± 0.08 ng/ml, n=4) in the media of cultured porcine TM cells that had been transfected with the human uPA gene, whereas no detectable uPA was found in the control cells. Conclusions: Cultured human TM cells taken from POAG patients were shown to express uPA. Further, gene transfer of uPA, which may degrade the extracellular matrix in juxta–canalicular connective tissue, into TM cells was possible. The present method may be applicable for chemical trabeculotomy procedures and may lead to development of a new therapy for open angle glaucoma.

Keywords: trabecular meshwork • extracellular matrix • outflow: trabecular meshwork