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.