April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Polarized Secretion of TGF-β and Thrombospondin from Human Fetal RPE
Author Affiliations & Notes
  • Louis K. Hirsch
    Doheny Vision Research Ctr, USC, Keck School of Medicine, Los Angeles, California
  • Parameswaran G. Sreekumar
    Doheny Vision Research Ctr, USC, Keck School of Medicine, Los Angeles, California
  • Christine Spee
    Doheny Vision Research Ctr, USC, Keck School of Medicine, Los Angeles, California
  • Danhong Zhu
    Doheny Vision Research Ctr, USC, Keck School of Medicine, Los Angeles, California
  • David R. Hinton
    Doheny Vision Research Ctr, USC, Keck School of Medicine, Los Angeles, California
  • Footnotes
    Commercial Relationships  Louis K. Hirsch, None; Parameswaran G. Sreekumar, None; Christine Spee, None; Danhong Zhu, None; David R. Hinton, None
  • Footnotes
    Support  California Institute for Regenerative Medicine DRI-01-444, NIH Core Grant EY03040, & Research to Prevent Blindness Grant
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 896. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : 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.

Methods: : (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.

Results: : 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.

Conclusions: : 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.

Keywords: retinal pigment epithelium • age-related macular degeneration • immunomodulation/immunoregulation 
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