April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
The Raf Pathway Mediates Insulin Stimulated VEGF Synthesis in Human Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • J. A. Conley
    Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, Michigan
  • P. C. Kothary
    Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, Michigan
  • M. A. Del Monte
    Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, Michigan
  • Footnotes
    Commercial Relationships  J.A. Conley, None; P.C. Kothary, None; M.A. Del Monte, None.
  • Footnotes
    Support  Skillman Foundation and Fight for Sight
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 5374. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      J. A. Conley, P. C. Kothary, M. A. Del Monte; The Raf Pathway Mediates Insulin Stimulated VEGF Synthesis in Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5374.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose: : Intensive insulin treatment often worsens diabetic retinopathy. Insulin stimulates vascular endothelial growth factor (VEGF) synthesis in human retinal pigment epithelial (hRPE) cells and this may play a role in diabetic retinopathy. Raf pathways are also involved in hRPE cell proliferation. However, the molecular mechanism in which insulin stimulates VEGF synthesis as well as how or if the Raf pathway is involved is unknown. This study evaluated the role of the Raf pathway in insulin stimulated VEGF synthesis.

Methods: : RPE primary cultures were obtained from human eyes from the Michigan Eye Bank. Cells were cultured to confluence in F12 medium supplemented with fetal bovine serum (FBS). Experimental reagents including insulin (0.5 - 5 ug/ml) and Raf inhibitor (Raf-I) (30 uM) plus insulin 5 ug/ml were added for 24-48 hours. Cellular proliferation was quantitated using 3H- thymidine incorporation as well as direct cell counting of viable cells using the trypan blue exclusion method. Immuno-precipitation of 14C-methionine VEGF (14C-VEGF) and immuno-cytochemistry were performed to study intracellular VEGF synthesis. Student "t" test was used to analyze the data, with p<0.05 considered significant.

Results: : Insulin stimulated 3H-thymidine incorporation and 14C-VEGF synthesis in a dose dependent manner. There was a decrease in 3H-thymidine incorporation with Raf-I plus insulin versus insulin alone (975.28 ± 25.62 vs. 1123.93 ± 84.15, CPM ± SEM, p<0.05, n=4) as well as a decrease in 14C-VEGF synthesis with Raf-I plus insulin versus insulin alone (1204.72 ± 345.18 vs. 2091.88 ± 270.34, CPM ± SEM, p<0.05, n=6). Immunoreactivity of VEGF increased in the presence of insulin (5 ug/ml) and decreased with insulin plus Raf-I, as shown by immuno-histochemical studies.

Conclusions: : This study demonstrates that insulin is a mitogen for hRPE cell proliferation and the Raf pathway mediates insulin-induced proliferation of hRPE cells. In addition, it demonstrates that insulin stimulates VEGF synthesis in hRPE cells using a mechanism mediated by the Raf pathway. Further elucidation of the role of the Raf pathway in VEGF synthesis may result in the development of potential new drugs for the treatment of diabetic retinopathy.

Keywords: vascular endothelial growth factor • diabetic retinopathy 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×