June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Inhibiting VEGF signaling by targeting receptor endocytosis
Author Affiliations & Notes
  • Weiquan Zhu
    Department of Medicine, University of Utah, Program in Molecular Medicine, Salt Lake City, UT
  • Dallas Shujing Shi
    University of Utah, Human Genetics, Salt Lake City, UT
    University of Utah, MD/PhD program, Salt Lake City, UT
  • Jacob M. Winter
    Department of Medicine, University of Utah, Program in Molecular Medicine, Salt Lake City, UT
  • Dean Li
    Department of Medicine, University of Utah, Program in Molecular Medicine, Salt Lake City, UT
    University of Utah, Human Genetics, Salt Lake City, UT
  • Footnotes
    Commercial Relationships Weiquan Zhu, None; Dallas Shi, None; Jacob Winter, None; Dean Li, Navigen LLC (C)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 233. doi:
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      Weiquan Zhu, Dallas Shujing Shi, Jacob M. Winter, Dean Li; Inhibiting VEGF signaling by targeting receptor endocytosis. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):233.

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

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Abstract
 
Purpose
 

Diabetic Retinopathy (DR) is a common microvascular complication of diabetes mellitus where excessive VEGF signaling through cognate cell surface receptor tyrosine kinases (VEGFRs) leads to endothelial cell proliferation and migration. VEGF-induced VEGFR internalization is essential for signal amplification, yet the mechanisms coordinating the trafficking and intracellular signaling of VEGFR remain unknown. Here, we hypothesize a small GTPAses, ARF6, is crucial for coordinating VEGF-induced VEGR2 internalization and signal amplification and may be a new therapeutic target for treating DR.

 
Methods
 

Using ARF6 siRNA or control siRNA in cultured human retinal endothelial cells (HRECs), we measured VEGF-induced HREC migration, VEGFR2 signaling, and VEGFR2 internalization. We determined which ARFGEFs mediates VEGF-induced ARF6 activation using siRNAs and co-IPs. Then, we used a high throughput fluorometric biochemical assay to identify chemically tractable, direct, reversible, allosteric inhibitors of ARF6. To test the utility of targeting ARF6 in DR pathologies, we used the new ARF6 inhibitors to inhibit VEGF-induced and streptozocin-induced retinal permeability in vivo.

 
Results
 

We show that VEGFR2 constitutively binds the guanine nucleotide exchange factor, GEP100, and in the presence of VEGF, the VEGFR-GEP100 complex activates ADP-ribosylation factor 6 (ARF6) (n=3, p<0.05). ARF6 activation promotes binding of VEGFR to its co-receptor, neuropilin, internalization of the receptor, and amplification of receptor tyrosine kinase (RTK) signaling cascades (n=3, p<0.05). We identify a small molecule inhibitor of ARF6 that blunts internalization of VEGFR, prevents its immediate downstream signaling, and mutes pathologic endothelial hyperpermeability in mouse models of diabetic retinopathy (n³3, p<0.05).

 
Conclusions
 

Our work highlights the critical role of ligand activated receptor endocytosis in signal amplification and the importance of the small GTPase, ARF6, as a mediator of this process. This presents a new target for treating excessive VEGF signaling in DR.  

 
Proposed VEGF-NRP1-GEP100-ARF6 signaling pathway
 
Proposed VEGF-NRP1-GEP100-ARF6 signaling pathway

 
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