PURPOSE: Retinal pigment epithelial (RPE) cells are known to play important roles in maintaining the homeostasis of the retina and in controlling choroidal neovascularization. The purpose of this study was to identify a factor or factors that would stimulate RPE cells to proliferate. METHODS: To isolate such a factor, 100 L of human-fibroblast-conditioned medium underwent ion-exchange, hydrophobic, and reverse-phase chromatographies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The growth-promoting activity of the factor was examined in a human K-1034 RPE cell line and human primary RPE cells. RESULTS: The different chromatographic processes isolated a 31-kDa factor that had RPE cell growth-promoting properties. This factor, which we have named RPE cell factor (REF)-1, promotes growth of RPE cells but not of human umbilical vein endothelial cells (HUVECs). The amino-terminal sequence and molecular cloned cDNA of REF-1 were identical with those of tissue-factor pathway inhibitor (TFPI)-2, a family of TFPIs, and placental protein (PP)-5, a serine protease inhibitor. The cDNA expression of REF-1/TFPI-2 with pcDL-pSRalpha vector in Chinese hamster ovary (CHO) cells confirmed the growth-promoting activity for RPE cells. The major component of the recombinant REF-1/TFPI-2 expressed in CHO cells had a molecular mass of 31 kDa and exerted growth-promoting activity in RPE cells but not in human endothelial cells and fibroblasts in vitro. REF-1/TFPI-2 also had protease inhibitory activity. The other family factor, TFPI-1, did not promote RPE cell growth. CONCLUSIONS: REF-1/TFPI-2 is a novel growth-promoting factor for RPE cells but not for endothelial cells and fibroblasts. Its properties make it potentially beneficial for intraocular therapy for the repair and maintenance of RPE cells.
PURPOSE: Retinal pigment epithelial (RPE) cells are known to play important roles in maintaining the homeostasis of the retina and in controlling choroidal neovascularization. The purpose of this study was to identify a factor or factors that would stimulate RPE cells to proliferate. METHODS: To isolate such a factor, 100 L of human-fibroblast-conditioned medium underwent ion-exchange, hydrophobic, and reverse-phase chromatographies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The growth-promoting activity of the factor was examined in a human K-1034 RPE cell line and human primary RPE cells. RESULTS: The different chromatographic processes isolated a 31-kDa factor that had RPE cell growth-promoting properties. This factor, which we have named RPE cell factor (REF)-1, promotes growth of RPE cells but not of human umbilical vein endothelial cells (HUVECs). The amino-terminal sequence and molecular cloned cDNA of REF-1 were identical with those of tissue-factor pathway inhibitor (TFPI)-2, a family of TFPIs, and placental protein (PP)-5, a serine protease inhibitor. The cDNA expression of REF-1/TFPI-2 with pcDL-pSRalpha vector in Chinese hamster ovary (CHO) cells confirmed the growth-promoting activity for RPE cells. The major component of the recombinant REF-1/TFPI-2 expressed in CHO cells had a molecular mass of 31 kDa and exerted growth-promoting activity in RPE cells but not in human endothelial cells and fibroblasts in vitro. REF-1/TFPI-2 also had protease inhibitory activity. The other family factor, TFPI-1, did not promote RPE cell growth. CONCLUSIONS:REF-1/TFPI-2 is a novel growth-promoting factor for RPE cells but not for endothelial cells and fibroblasts. Its properties make it potentially beneficial for intraocular therapy for the repair and maintenance of RPE cells.
Authors: Grace E Lidgerwood; Anne Senabouth; Casey J A Smith-Anttila; Vikkitharan Gnanasambandapillai; Dominik C Kaczorowski; Daniela Amann-Zalcenstein; Erica L Fletcher; Shalin H Naik; Alex W Hewitt; Joseph E Powell; Alice Pébay Journal: Genomics Proteomics Bioinformatics Date: 2020-12-08 Impact factor: 7.691