PURPOSE: To investigate the alterations in gene expression when human retinal pigment epithelial (RPE) cells in culture are treated with vitreous as a model for the changes that occur in proliferative vitreoretinopathy. METHODS: Human RPE cells were cultured with or without human vitreous or collagen. RNA was extracted and reverse transcribed. The RNAs expressed were compared by using DNA macroarrays. Messenger RNA levels were also measured using real-time reverse transcription polymerase chain reaction. Protein expression was examined by immunoblot analysis. Immunoassays were used to determine levels of prostaglandin E(2). RESULTS: Vitreous treatment of RPE cells resulted in increased expression of two critical enzymes in the synthesis of prostaglandin E(2): membrane-associated prostaglandin E-synthase (mPGES) and cyclooxygenase (COX)-2. Increased levels of mPGES RNA and protein were still present at 48 hours of treatment, but the increase in COX-2 mRNA and protein was transient. The increase in the expression of mPGES was associated with an increase in the production of prostaglandin E(2) that was observed at 12 and 24 hours of treatment but not at 48 hours. Treatment with 100 microg collagen I per ml medium did not cause increased expression of mPGES and COX-2, even though both collagen- and vitreous-treatment caused a morphologic change in the RPE cells to a more fibroblast-like phenotype. CONCLUSIONS: Treatment of human RPE cells with vitreous induces changes in gene expression that are indicative of an inflammatory response.
PURPOSE: To investigate the alterations in gene expression when human retinal pigment epithelial (RPE) cells in culture are treated with vitreous as a model for the changes that occur in proliferative vitreoretinopathy. METHODS:Human RPE cells were cultured with or without humanvitreous or collagen. RNA was extracted and reverse transcribed. The RNAs expressed were compared by using DNA macroarrays. Messenger RNA levels were also measured using real-time reverse transcription polymerase chain reaction. Protein expression was examined by immunoblot analysis. Immunoassays were used to determine levels of prostaglandin E(2). RESULTS:Vitreous treatment of RPE cells resulted in increased expression of two critical enzymes in the synthesis of prostaglandin E(2): membrane-associated prostaglandin E-synthase (mPGES) and cyclooxygenase (COX)-2. Increased levels of mPGES RNA and protein were still present at 48 hours of treatment, but the increase in COX-2 mRNA and protein was transient. The increase in the expression of mPGES was associated with an increase in the production of prostaglandin E(2) that was observed at 12 and 24 hours of treatment but not at 48 hours. Treatment with 100 microg collagen I per ml medium did not cause increased expression of mPGES and COX-2, even though both collagen- and vitreous-treatment caused a morphologic change in the RPE cells to a more fibroblast-like phenotype. CONCLUSIONS: Treatment of human RPE cells with vitreous induces changes in gene expression that are indicative of an inflammatory response.
Authors: Ren Hartung; Sunil K Parapuram; Ramapriya Ganti; D Margaret Hunt; Kakarla V Chalam; Richard C Hunt Journal: Mol Vis Date: 2007-01-24 Impact factor: 2.367