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Y Tanaka, J Utsumi, T Sudo, N Nakamura, K Kigasawa, T Iwata, M Matsui; Purification, Molecular Cloning and Expression of A Novel Growth Promotive Factor for Retinal Pigment Epithelial Cells, REF-1/TFPI-2 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4583.
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Purpose: Retinal pigment epithelial (RPE) cells are known to play important roles to maintain homeostasis of the retina and to control choroidal neovascularization. The factor which stimulates RPE cell growth should be very valuable for treatment of some retinal diseases. The purpose of this study is to discover a specific growth promotive factor for RPE cells. Methods: One hundred liter of human fibroblast conditioned medium was applied to ion-exchange, hydrophobic and reversed-phase chromatographies and sodium dodecyl sulphate-polyacrylamide gel electrophoresis to purify RPE cell growth promotive activity. Human K-1034 RPE cells for the activity determination were originally established as reported earlier (Kigasawa et al.Jpn. J. Ophthalmol. 38, 10-15, 1994). Human endothelial cells were separated from human umbilical veins. Cell growth activity was determined by cultivation of test cells with samples from chromatographies. Molecular cloning was performed from placental cDNA library. The expression vector of cloned cDNA of the growth promotive factor was constructed with pSRαvector and transfected to Chinese ovary (CHO) cells. The expressed recombinant protein was purified and characterized. Results: We found and isolated 31kDa factor that has growth promotive activity for RPE cells but not for human endothelial cells (REF-1: RPE cell factor-1). The amino-terminal sequence of REF-1 was identical to that of tissue-factor pathway inhibitor-2 (TFPI-2), a family of TFPIs or placental protein 5 (PP5), a serine protease inhibitor. The cDNA of REF-1/TFPI-2 was cloned from placental cDNA library and expressed in CHO cells. Recombinant REF-1/TFPI-2 was 31kDa as a major component and expressed a growth promotive activity for RPE cells but not for endothelial cells and has protease inhibitory activity in vitro. The other family factor, TFPI-1 did not promote RPE cell growth. Conclusion: We found a novel activity of TFPI-2 as a growth promotive factor for RPE cells, REF-1. Because REF-1/TFPI-2 did not stimulate endothelial cells, the factor may have beneficial effect for the repair and maintenance of RPE cells membrane in vivo.
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