May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
The Role of Ras and Raf in FBS-Stimulated hRPE Cell Proliferation and VEGF Synthesis
Author Affiliations & Notes
  • D. M. Louie
    Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan
  • P. C. Kothary
    Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan
  • M. A. Del Monte
    Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan
  • Footnotes
    Commercial Relationships  D.M. Louie, None; P.C. Kothary, None; M.A. Del Monte, None.
  • Footnotes
    Support  Skillman Foundation
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 3541. doi:https://doi.org/
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      D. M. Louie, P. C. Kothary, M. A. Del Monte; The Role of Ras and Raf in FBS-Stimulated hRPE Cell Proliferation and VEGF Synthesis. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3541. doi: https://doi.org/.

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

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Abstract

Purpose: : Human retinal pigment epithelial (hRPE) cell proliferation and VEGF expression both play key roles in the pathogenesis of blinding proliferative retinal diseases. Prior studies have shown that the Ras/Raf/MEK/ERK pathway is important in the regulation of hRPE cell proliferation. However, little is known regarding the signaling pathway leading to VEGF synthesis in hRPE cells. We investigated the involvement of Ras and Raf in VEGF synthesis by cultured hRPE cells.

Methods: : Primary hRPE cell cultures were established from post mortem human eyes. FBS (10%) stimulated hRPE cell proliferation in the presence of either Mevastatin, an inhibitor of Ras, or GW5074, an inhibitor of Raf, was measured using both 3H-thymidine incorporation and the trypan blue exclusion methods. The effect of Mevastatin and GW5074 on FBS (10%) stimulated VEGF synthesis by hRPE was then determined by measuring immunoprecipitated 14-C-methionine-VEGF. Localization of intracellular VEGF was performed using immunohistochemistry. Data were analyzed by Student 't' test.

Results: : FBS stimulated hRPE cell proliferation in a dose dependent manner. Inhibition of either Ras or Raf led to the marked inhibition of FBS stimulated hRPE cell proliferation (71% and 89% inhibition, respectively) as measured by 3H-thymidine incorporation. Cell counts using the trypan blue exclusion method likewise showed reduction of hRPE cell proliferation after incubation with FBS and Mevastatin or FBS and GW5074, as compared to FBS stimulated controls. Measurements of immunoprecipitated 14-C-methionine-VEGF showed inhibition of VEGF synthesis by 40% and 78% with Ras and Raf inhibition, respectively. In addition, immunohistochemical studies demonstrated qualitatively less nuclear VEGF positive immunoreactivity in FBS stimulated hRPE cells exposed to Ras/Raf inhibitors when compared to FBS stimulated control cells.

Conclusions: : These results are consistent with prior studies which implicate the role of the Ras/Raf signaling pathway in hRPE cell proliferation, and suggest that a key role of Ras/Raf signaling in hRPE VEGF synthesis may be the mechanism. Ras/Raf inhibition may therefore prove useful in the treatment of proliferative retinal diseases.

Keywords: retinal pigment epithelium • proliferative vitreoretinopathy • vascular endothelial growth factor 
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