Targeting ARF6 to Switch off Inflammatory Vascular Hyper-permeability of Blood-Retina Barrier
Helong Zhao1, Dallas S. Shi1, Jacob M. Winter1, Bianca E. Rich1, Zongzhong Tong2, Lise K. Sorensen1, Yi Huang1, Jing Ling1, Jake A. Bergquist1, Jackson R. Richards1, Amanda Jiang1, M. Elizabeth Hartnett1, Alan L. Mueller2, Kirill Ostanin2, Kirk R. Thomas1, Shannon J. Odelberg1, Dean Y. Li1 and Weiquan Zhu1
1Program in Molecular Medicine, University of Utah, Salt Lake City, Utah, USA; 2Navigen Inc., Salt Lake City, Utah, USA
Blood-Retina Barrier (BRB) is a unique component of the Blood-CNS Barrier (BCNSB). Similar to Blood-Brain Barrier (BBB), the inner BRB is maintained by dense cell-cell junctions of endothelium that protects retina and optical nerves from interference from blood content. Neuronal inflammation in diabetic and other retinopathies are associated with BRB destabilization induced by inflammatory cytokines, including VEGF and IL-1β.
We previous identified a small GTPase - ARF6 - that functions as the nodal controlling switch for many inflammatory cytokine signaling pathways that cause pathological endothelial hyper-permeability, including VEGF, IL-1β, and LPS [Nature Cell Biology, 2009; Nature, 2012; JI, 2014; JCI, 2017]. In this study, we show:
Inflammatory retinal pathologies (diabetes and pathogen-induced) are associated with increased ARF6 activity in retinal endothelium: We discovered that the inflamed eye tissues of human diabetic patients have upregulated ARF6 expression. In two retinal inflammation mouse models, diabetes (STZ model) induced inflammation and LPS induced inflammation increased ARF6 activation in mouse retina. Corresponding inflammatory cytokines in these two retinal pathologies, VEGF and IL-1β, also activated ARF6 in human retinal endothelial cells.
ARF6 activation is necessary for inflammatory hyper-permeability of retinal endothelium: When inhibiting ARF6 in human retinal endothelial monolayer cultures using ARF6 siRNA or ARF6 inhibitor (NAV-2729), VEGF and IL-1β induced endothelial hyper-permeability was prevented. Mechanistically, ARF6 inhibition prevented the cell-cell junction (VE-Cadherin) breakdown following VEGF and IL-1β treatment.
Genetic and pharmacological targeting of ARF6 ameliorates inflammatory hyper-permeability of BRB in diabetes and pathogen-induced retinopathy: Using genetic endothelial Arf6 depletion (Tie2-Cre) and small molecular ARF6 inhibitor (NAV-2729), we showed that targeting ARF6 prevented BRB leakage (Evans Blue assay) and vascular malformation in diabetes (STZ) and hypoxia. Targeting ARF6 also prevented the in vivo retinal endothelium hyper-permeability induced by i.v. injected VEGF and IL-1β.