March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
The small GTPase Rap1 regulates intracellular ROS generation in RPE
Author Affiliations & Notes
  • Haibo Wang
    John A Moran Eye Ctr, Ophthalmology, University of Utah, Salt Lake City, Utah
  • Erika Wittchen
    Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina
  • M. Elizabeth Hartnett
    John A Moran Eye Ctr, Ophthalmology, University of Utah, Salt Lake City, Utah
  • Footnotes
    Commercial Relationships  Haibo Wang, None; Erika Wittchen, None; M. Elizabeth Hartnett, None
  • Footnotes
    Support  R01 R01EY015130 MEH
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 4277. doi:
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      Haibo Wang, Erika Wittchen, M. Elizabeth Hartnett; The small GTPase Rap1 regulates intracellular ROS generation in RPE. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4277.

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

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Abstract

Purpose: : We have previously shown that NADPH oxidase-mediated generation of reactive oxygen species (ROS) is involved in pathologic steps of neovascular AMD. Activation of NADPH oxidase involves assembly of the cytosolic (p47phox, p67phox, and Rac1) and membrane-associated components (NOX and p22phox). We recently showed that activation of a GTPase, Rap1, reduced CNV size in a laser injury model. We now tested the hypothesis that inhibition of Rap1 increases ROS generation in RPE cells by interfering with NADPH oxidase subunit assembly and activation.

Methods: : Using CM-H2DCFDA, intracellular ROS generation was measured in RPE cells infected with an adenoviral vector expressing GTPase-activating proteins (Ad-RapGAP) to inhibit Rap1 activity or control vector expressed with GFP (Ad-GFP). Protein interaction of Rap1 and NADPH oxidase subunit, p22Phox, in RPE cells was determined by co-immunoprecipitation of Rap1 and p22phox in RPE cells infected with the Ad-RapGAP or control Ad-GFP. To activate Rap1, RPE were exposed to an Epac-specific cAMP analogue, 8-pCPT-2_OMe-cAMP (8-CPT, Tocris Bioscience). Statistics were performed using ANOVA.

Results: : Compared with control Ad-GFP, ROS generation was increased in RPE cells infected with the Ad-RapGAP (P=0.003) and decreased in RPE cells incubated with 8-CPT (P=3.3464E-15). Compared to RPE infected with control Ad-GFP without 8-CPT treatment, infection of RPE with Ad-RapGAP decreased endogenous Rap1 bound to p22phox (P=0.000036), while incubation of RPE with 8-CPT restored the binding affinity of endogenous Rap1 to p22phox.

Conclusions: : Activated Rap1 prevented ROS generation in RPE cells. The molecular mechanism may involve inhibition of NADPH oxidase activation by interfering with NADPH oxidase subunit assembly.

Keywords: retinal pigment epithelium • cell adhesions/cell junctions • oxidation/oxidative or free radical damage 
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