May 2004
Volume 45, Issue 13
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ARVO Annual Meeting Abstract  |   May 2004
CHANGES IN mRNA EXPRESSION AND PROTEIN LEVEL OF ANGIOGENIC AND ANTI–ANGIOGENIC FACTORS IN CULTURED HUMAN FETAL RETINAL PIGMENT EPITHELIUM (hfRPE)
Author Affiliations & Notes
  • F.E. Wang
    National Eye Institute, National Institutes of Health, Bethesda, MD
    School of Optometry, University of California, Berkeley, Berkeley, CA
  • A. Jafari
    National Eye Institute, National Institutes of Health, Bethesda, MD
    School of Optometry, University of California, Berkeley, Berkeley, CA
  • G. Shi
    National Eye Institute, National Institutes of Health, Bethesda, MD
    School of Optometry, University of California, Berkeley, Berkeley, CA
  • K.G. Rendahl
    Gene Therapy Group, Chiron Corporation, Emeryville, CA
  • W.C. Manning
    Gene Therapy Group, Chiron Corporation, Emeryville, CA
  • M. Coyne
    Gene Therapy Group, Chiron Corporation, Emeryville, CA
  • S.S. Miller
    National Eye Institute, National Institutes of Health, Bethesda, MD
    School of Optometry, University of California, Berkeley, Berkeley, CA
  • Footnotes
    Commercial Relationships  F.E. Wang, Chiron Corporation F; A. Jafari, Chiron Corporation F; G. Shi, Chiron Corporation F; K.G. Rendahl, Chiron Corporation E; W.C. Manning, Chiron Corporation E; M. Coyne, Chiron Corporation E; S.S. Miller, Chiron Corporation F.
  • Footnotes
    Support  NIH EY02205, Core Grant EY03176, Foundation Fighting Blindness
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3711. doi:
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      F.E. Wang, A. Jafari, G. Shi, K.G. Rendahl, W.C. Manning, M. Coyne, S.S. Miller; CHANGES IN mRNA EXPRESSION AND PROTEIN LEVEL OF ANGIOGENIC AND ANTI–ANGIOGENIC FACTORS IN CULTURED HUMAN FETAL RETINAL PIGMENT EPITHELIUM (hfRPE) . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3711.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: Evaluate the interactions of vascular endothelial growth factor (VEGF), pigment–epithelium derived factor (PEDF), and sFlt1, each overexpressed in hfRPE, on their mRNA and protein levels. Methods: Primary human fetal RPE cells were seeded on transwell plate coated with human extracellular matrix. After cells were confluent and differentiated, they were infected with AAV–VEGF, AAV–PEDF or AAV–sFlt1 with AAV–GFP as control. HfRPE conditioned medium was collected from apical and basal compartments every 2–3 days for ELISA over a three–week period. Total RNA was extracted for quantitative reverse transcription polymerase chain reaction (Q–RT–PCR). Results: VEGF mRNA increased 2, 4, or 270 fold in hfRPE infected with AAV–PEDF, AAV–sFlt1, or AAV–VEGF, respectively. Apical VEGF protein decreased 20%–40% (p<0.05) in AAV–sFlt1 infected cells and increased 2–3 times (p<0.05) in AAV–VEGF infected cells while the VEGF level on basal side did not change significantly in both groups. In AAV–PEDF infected cells, VEGF protein did not significantly change in either the apical or basal compartments. In hfRPE infected with AAV–PEDF, AAV–sFlt1, or AAV–VEGF, PEDF mRNA increased 6, 2, or 2 fold, respectively. In hfRPE infected with AAV–sFlt1, the sFlt1 protein increased more than 100–fold in the apical compartment and more than 10–fold in the basal compatment(n= 2). The sFlt1 protein increased in AAV–VEGF infected samples and decreased in AAV–PEDF infected samples. Conclusions: Overexpression of PEDF in hfRPE can increase VEGF mRNA expression with no significant changes in VEGF protein level. Overexpression of sFlt1 increased VEGF mRNA and decreased VEGF protein level on the apical side of the tissue. The decrease in apical VEGF may have been caused by a decrease in protein synthesis, but the concomitant increase in VEGF mRNA suggests otherwise. Alternatively, the decrease of apical VEGF protein, as measured by ELISA, may have been caused by increased sFlt1 protein secretion and binding to VEGF. The in vitro PEDF and sFlt1 data show that increased expression of anti–angiogenic factors do not increase VEGF protein level in either the apical or basal compartments. These results suggest that the RPE may differentially affect blood vessel proliferation in the extracellular spaces on the retinal and choroidal sides of the tissue.

Keywords: retinal pigment epithelium • gene transfer/gene therapy • choroid: neovascularization 
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