Norbert Kociok1, Antonia M Joussen. 1. Department of Ophthalmology, Heinrich Heine University, Duesseldorf, Germany. norbert.kociok@charite.de
Abstract
BACKGROUND: RPE cells are a major player in various diseases of the retina and choroid. Proliferating RPE cells are thought to be an initiating factor in proliferative vitreoretinopathy (PVR); the aging RPE cells are important in age-related macular degeneration (AMD). Early passages of cultured human retinal pigment epithelial cells were used as a model system to identify differentially expressed genes in proliferating retinal pigment epithelial (RPE) cells. METHODS: A differential expression analysis (DEmRNA-PCR) was used to find differentially expressed mRNA in early passages of cultured human RPE cells. The detected mRNAs were identified by sequencing. Their differential expression was verified by semi-quantitative RT-PCR. The expression of the identified protein in vitro and its presence in surgically removed epiretinal membranes was demonstrated by western blotting and immunocytochemical analysis. RESULTS: DEmRNA-PCR detected a decreased expression of a band at approximately 530 bp in human RPE cells of passage 3 (P3) compared to P0. This band was identified as part of the human complement regulatory factor H, a cofactor to complement factor I. The mRNA expression of both regulatory proteins of the complement system was confirmed in freshly prepared human RPE cells and in cultured cells from P0 to P8. The protein expression was verified in cultured RPE cells. The expression of both proteins in surgically removed epiretinal membranes was demonstrated by immunohistochemistry. CONCLUSION: The identification of the differential expression of the regulatory factors H and I of the complement system in cultured RPE cells by a technique without any prerequisites demonstrates and confirms the importance of these factors in RPE cells. In addition to its known role in age-related macular degeneration, the presence of these complement factors in epiretinal membranes may also indicate a role of the complement system in proliferative retinopathy.
BACKGROUND: RPE cells are a major player in various diseases of the retina and choroid. Proliferating RPE cells are thought to be an initiating factor in proliferative vitreoretinopathy (PVR); the aging RPE cells are important in age-related macular degeneration (AMD). Early passages of cultured human retinal pigment epithelial cells were used as a model system to identify differentially expressed genes in proliferating retinal pigment epithelial (RPE) cells. METHODS: A differential expression analysis (DEmRNA-PCR) was used to find differentially expressed mRNA in early passages of cultured human RPE cells. The detected mRNAs were identified by sequencing. Their differential expression was verified by semi-quantitative RT-PCR. The expression of the identified protein in vitro and its presence in surgically removed epiretinal membranes was demonstrated by western blotting and immunocytochemical analysis. RESULTS: DEmRNA-PCR detected a decreased expression of a band at approximately 530 bp in human RPE cells of passage 3 (P3) compared to P0. This band was identified as part of the human complement regulatory factor H, a cofactor to complement factor I. The mRNA expression of both regulatory proteins of the complement system was confirmed in freshly prepared human RPE cells and in cultured cells from P0 to P8. The protein expression was verified in cultured RPE cells. The expression of both proteins in surgically removed epiretinal membranes was demonstrated by immunohistochemistry. CONCLUSION: The identification of the differential expression of the regulatory factors H and I of the complement system in cultured RPE cells by a technique without any prerequisites demonstrates and confirms the importance of these factors in RPE cells. In addition to its known role in age-related macular degeneration, the presence of these complement factors in epiretinal membranes may also indicate a role of the complement system in proliferative retinopathy.
Authors: Robert J Klein; Caroline Zeiss; Emily Y Chew; Jen-Yue Tsai; Richard S Sackler; Chad Haynes; Alice K Henning; John Paul SanGiovanni; Shrikant M Mane; Susan T Mayne; Michael B Bracken; Frederick L Ferris; Jurg Ott; Colin Barnstable; Josephine Hoh Journal: Science Date: 2005-03-10 Impact factor: 47.728
Authors: Jonathan L Haines; Michael A Hauser; Silke Schmidt; William K Scott; Lana M Olson; Paul Gallins; Kylee L Spencer; Shu Ying Kwan; Maher Noureddine; John R Gilbert; Nathalie Schnetz-Boutaud; Anita Agarwal; Eric A Postel; Margaret A Pericak-Vance Journal: Science Date: 2005-03-10 Impact factor: 47.728