Controllable Urokinase Gene Expression in Trabecular Meshwork Cells
|Mei Tsuda1*, Shiho Kaneko1, Akira Ando1, Tetsuya Nishimura1, Emiko Okuda-Ashitaka2, Seiji Ito2, Makoto Taomoto3, Miyo Matsumura3 and Kanji Takahashi3|
|1Department of Ophthalmology, Kansai Medical University Takii Hospital, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan|
|2Department of Medical Chemistry, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8506, Japan|
|3Department of Ophthalmology, Kansai Medical University Hirakata Hospital, 2-3-1 Shin-machi, Hirakata, Osaka 573-1191, Japan|
|Corresponding Author :||Mei Tsuda, M.D
Department of Ophthalmology
Kansai Medical University Takii Hospital
10-15 Fumizono-cho, Moriguchi, Osaka 570-8507,Japan
Tel: +81-6-6992-1001 ext.3324
E-mail: [email protected]
|Received: December 19, 2013; Accepted: March 11, 2014; Published: March 17, 2014|
|Citation: Tsuda M, Kaneko S, Ando A, Nishimura T, Okuda-Ashitaka E, et al. (2014) Controllable Urokinase Gene Expression in Trabecular Meshwork Cells. J Clin Exp Ophthalmol 5:329. doi:10.4172/2155-9570.1000329|
|Copyright: © 2014 Tsuda M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.|
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Purpose: Glaucoma is a type of progressive optic neuropathy that finally leads to blindness related to elevated intraocular pressure (IOP). Accumulation of extracellular matrix in trabecular meshwork (TM) and juxta-canalicular connective tissues, which form an aqueous outflow pathway, may be a major cause of increased IOP, thus the fibrinolytic system may be associated with regulation of IOP. We examined the possibility of controllable urokinase plasminogen activator (uPA) gene transfer into TM cells.
Methods: TM cells were freshly isolated from porcine eyes and human TM cells obtained during trabeculectomy procedures and cultured. Total RNA was extracted from human TM cells and reverse transcribed into cDNA. A reverse-transcribed polymerase chain reaction (RT-PCR) method was then performed to detect the gene expression of uPA. The cDNA of human uPA was sub-cloned into an expression vector (pEYFP-N1 vector) and controllable expression vector (TRE-Tight vector), then each vector was independently transfected into cultured porcine TM cells. Doxycycline was added to the culture medium to activate the pTet-ON and TRE-Tight vectors. Finally, the expression of uPA was examined using an enzyme linked immunosorbent assay (ELISA).
Results: ELISA findings revealed the expression of uPA (3.5 ng/ml) in medium from cultured porcine TM cells that had been transfected with the human uPA gene using the pEYFP-N1 vector. Doxycycline induced human uPA in the pTet-On and TRE-Tight vectors with human uPA gene co-transfected TM cells in a dose-dependent manner.
Conclusions: Controllable gene transfer of uPA, which may degrade the extracellular matrix in juxta-canalicular connective tissue, into TM cells was achieved using a Tet-On system. The present method may be useful as a novel therapy for glaucoma.