Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy
Weiquan Zhu, … , Shannon J. Odelberg, Dean Y. Li
Weiquan Zhu, … , Shannon J. Odelberg, Dean Y. Li
Published October 23, 2017
Citation Information: J Clin Invest. 2017;127(12):4569-4582. https://doi.org/10.1172/JCI91770.
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Research Article Cell biology Ophthalmology

Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy

Abstract

The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs — ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy.

Authors

Weiquan Zhu, Dallas S. Shi, Jacob M. Winter, Bianca E. Rich, Zongzhong Tong, Lise K. Sorensen, Helong Zhao, Yi Huang, Zhengfu Tai, Tara M. Mleynek, Jae Hyuk Yoo, Christine Dunn, Jing Ling, Jake A. Bergquist, Jackson R. Richards, Amanda Jiang, Lisa A. Lesniewski, M. Elizabeth Hartnett, Diane M. Ward, Alan L. Mueller, Kirill Ostanin, Kirk R. Thomas, Shannon J. Odelberg, Dean Y. Li

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Figure 6

GEP100 activates ARF6 to promote binding of VEGFR2 and NRP1, which regulates VEGFR2 intracellular trafficking and signal output.

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GEP100 activates ARF6 to promote binding of VEGFR2 and NRP1, which regul...
(A) ARF6 siRNA– or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes, VEGFR2 was immunoprecipitated, and the precipitate was immunoblotted using antibodies against NRP1. Groupings of images are different parts of the same gel. (B) ARNO siRNA–, GEP100 siRNA–, or control siRNA–treated HRMECs were stimulated with VEGF for 5 minutes and assayed for VEGFR2-NRP1 coimmunoprecipitation as described for A. (C) GEP100 siRNA– or control siRNA-treated HRMECs were infected with AdARF6Q67L for 36 hours and then stimulated with VEGF for 5 minutes and assayed for VEGFR2-NRP1 coimmunoprecipitation as described for A. (D) Pitstop 2–treated and/or GEP100 siRNA– or control siRNA–transfected HRMECs were stimulated with VEGF for 5 minutes and assayed for VEGFR2/NRP1 coimmunoprecipitation as described for A. (E) HRMECs were stimulated with VEGF for 5 minutes and assayed by immunofluorescence to determine the intracellular location of ARNO and GEP100. Scale bars: 30 μm; original magnification, ×1,200. In A–D, a 1-way ANOVA with Tukey’s multiple comparisons tests was used to assess statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). All error bars represent the SEM. n = 3 for all panels.