Vitreous modulation of gene expression in low-passage human retinal pigment epithelial cells.
OBJECTIVE
In proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cells enter the vitreous and proliferate. They become fibroblast-like and participate in the formation of contractile membranes, which can lead to retinal detachment. Vitreous treatment of RPE cells in vitro results in similar morphologic changes. This study was conducted to examine vitreous-induced modulation of gene expression in RPE cells.


METHODS
Low-passage human RPE cell lines derived from three donors were each treated for 6, 12, 24, or 48 hours with complete medium or complete medium containing 25% vitreous. Changes in mRNA levels were examined by using microarrays. Real-time quantitative PCR (qPCR) was used to measure mRNA expression of a subset of genes in cells from three additional donors. Immunohistochemistry and immunoblot analysis were used to examine protein expression.


RESULTS
Vitreous treatment caused a progressive reprogramming of gene expression. qPCR confirmed vitreous modulation of mRNA levels of 10 of 10 genes. Changes consistent with a transition from an epithelial to a mesenchymal phenotype were observed. Downregulated genes included genes associated with differentiated RPE cells. Upregulated genes included genes associated with stress and inflammation. Pathway analysis indicated that the transforming growth factor-beta/bone morphogenetic protein (BMP) pathway and the focal adhesion pathway may play a role in this process. BMP-2 protein and mRNA were increased.


CONCLUSIONS
Despite the biological variation in vitreous and RPE donors, vitreous reproducibly modulated a limited number of mRNAs. Many of these changes were consistent with the more fibroblast-like appearance of vitreous-treated cells and with the pathobiology of PVR. TGF-beta and BMP-2 may be important modulators of vitreous-induced changes in gene expression.
Ganti
Ramapriya
R
Departments of Pathology, Microbiology and Immunology, School of Medicine, University of South Carolina, Columbia, SC 29208, USA.
Hunt
Richard C
RC
Parapuram
Sunil K
SK
Hunt
D Margaret
DM
D002478:Cells, Cultured; D005136:Eye Proteins / Q000235:genetics*; D015870:Gene Expression / Q000502:physiology*; D020869:Gene Expression Profiling; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D020411:Oligonucleotide Array Sequence Analysis; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D014822:Vitreous Body / Q000502:physiology*
D005136:Eye Proteins; D012333:RNA, Messenger