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Louis K. Hirsch, Parameswaran G. Sreekumar, Christine Spee, Danhong Zhu, David R. Hinton; Polarized Secretion of TGF-β and Thrombospondin from Human Fetal RPE. Invest. Ophthalmol. Vis. Sci. 2011;52(14):896.
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Retinal pigmented epithelium (RPE) secretes fibrogenic proteins, e.g. transforming growth factor β1 & β2 (TGF-β1 & TGF-β2), and thrombospondin-1 (TSP-1). These proteins contribute to the pathogenesis of macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE cell polarity may be altered in AMD and PVR, we studied effects of polarity on protein secretion.
(1) Human Fetal RPE (Hf-RPE) culture: RPE from 2 donors were cultured under 3 conditions for comparison: subconfluent nonpolarized, confluent nonpolarized, and highly polarized monolayers. (2) Enzyme-linked immunosorbent assay (ELISA): TGF-β1, TGF-β2, and TSP-1 were measured in media from apical and basal compartments of polarized RPE transwell filters and from the single compartment of nonpolar RPE petri dishes. (3) Transepithelial Resistance(TER): To confirm RPE differentiation, donors had TERs of 235 & 185 Ω•cm^2. (4) Normalization: Data normalized by total cell protein assays.
Comparing polarized and nonpolarized hf-RPE, nonpolarized cells secreted higher concentrations of TGF-β2 in both active and inactive isoforms. Subconfluent and confluent nonpolarized RPE secreted 21-fold (p<0.001) and 399-fold (p<0.001) greater concentrations respectively. Comparing apical and basal secretions from polarized hf-RPE, TGF-β2 was secreted in 5-fold greater concentrations apically (p = 0.004). TGF-β1 was secreted in 6-fold greater concentrations basally. TSP-1 was secreted equally in both compartments.
Nonpolarized RPE secretes higher concentrations of TGF-β2 than polarized RPE. Additionally, differentiated RPE secretes TGF-β2 & TGF-β1 with polar distribution. These data suggest that protein over-secretion, and altered protein distribution, contribute to pathogenesis in disorders with altered RPE polarity, e.g. AMD and PVR.
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