Abstract
Purpose :
Diabetic retinopathy (DR) is a common microvascular complication of diabetes mellitus and is one of the fastest-growing causes of blindness and visual impairment in the working-age population. Breakdown of the blood-retinal barrier is a common pathologic feature in diabetic patients and experimental animal models. The dominant emphasis in the field has been studying the effects of angiogenic and proinflammatory cytokines destabilizing the endothelial barrier. This focus has shaped our current therapeutic strategies, which primarily ameliorate destabilizing pathways by blocking VEGF or proinflammatory cytokine signaling. However, an alternative approach that focuses on strengthening pathways that promote vascular stability, for example, angiopoietin-1 (ANG1)/TIE2, has recently begun to gain traction. Here, we show that inhibition of a small GTPase ARF6 by ANG-1 receptor TIE2 contributes to this stabilization via the activation of the ARF GTPase-activating protein (ARFGAP) GIT1, which converts activated ARF6 to its inactive form.
Methods :
The barrier function of human retinal microvascular endothelial cells (HRMVECs) monolayer was measured by transendothelial electrical resistance (TEER) following the treatment with VEGF with or without ANG1 or with the expression of constitutively active ARF6Q67L. In addition, endothelial-specific Git1 knockout mice were used to test dermal and retinal vascular permeability induced by VEGF combined with or without the treatment with ANG-1 or ARF6 inhibitor NAV-2729.
Results :
Our results suggest that an ANG1-TIE2-GIT1 cascade inactivates ARF6. ANG1 can basally enhance endothelial barrier function and inhibit VEGF-induced endothelial permeability in HRMVECs. However, ANG1 cannot enhance barrier function measured by transendothelial electrical resistance (TEER) in monolayers transduced with adenoviruses expressing constitutively active ARF6Q67L. Our data also suggest that both VEGF and loss of GIT1 induce ARF6 activation and permeability in endothelial cells. Furthermore, the synergistic effects of VEGF stimulation and loss of GIT1 expression in mouse retinal permeability can be reversed by the ARF6 inhibitor NAV-2729 in vivo.
Conclusions :
Using in vitro, cellular, genetic, and pharmacologic techniques, we suggest that ANG1 function through GIT1 to inhibit ARF6, stabilizing the endothelium and preventing the vascular leak.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.