Adenosine deaminase-2-induced permeability increase in HRECs is suppressed by miR-146b-3p

Abstract

Purpose : Diabetic retinopathy (DR), a neuroinflammatory disease, includes excessively increased cytokine release, microglia activation and blood–retinal barrier (BRB) breakdown. The molecular mechanism of neuroinflammation leading to BRB breakdown remains unclear. Adenosine deaminase-2 (ADA2), important for macrophage proliferation, is negatively regulated by miR-146b-3p. This is an initial study to investigate the role of miR-146b-3p and ADA2 in BRB breakdown.

Methods : Human monocytes U937 were differentiated to macrophages by phorbol 12-myristate 13-acetate (PMA 50ng/mL). Cells were then treated with amadori glycated albumin (AGA 500 µg/mL) for 12 hour. ADA2 activity in the macrophages conditioned medium (CM) and in the vitreous of diabetic and non-diabetic, non-inflammatory human donor eyes was colorimetrically measured. TNFα and IL-6 were measured in macrophages CM by enzyme-linked immunosorbent assay (ELISA). Human retinal endothelial cells (HRECs) were treated with AGA, CM of AGA-treated macrophages, and CM of AGA-treated and miR-146b-3p-transfected macrophages and BRB of HRECs was evaluated by electric cell-substrate impedance sensing (ECIS) that measures the changes in transcellular electrical resistance (TER). ZO-1 distribution in HRECs was examined by immunofluorescence.

Results : Our results illustrated that ADA2 activity was significantly increased in the diabetic humans as compared with the control (p<0.05). ADA2 activity, TNFα and IL-6 levels were increased significantly in the CM of AGA-treated macrophages as compared with the control (p<0.001). ADA2 activity, TNFα and IL-6 levels were significantly decreased by miR-146b-3p (p<0.05). TER of HRECs was significantly decreased by CM of AGA-treated macrophages as compared with AGA treatment (p<0.0001). This effect was significantly attenuated by miR-146b-3p, but not control, transfection (p<0.001). Lastly, ZO-1 pattern in HRECs was significantly disturbed by CM of AGA-treated macrophages as compared with AGA treatment. This effect was attenuated by miR-146b-3p transfection.

Conclusions : Our results suggest that ADA2 and miR-146b-3p contribute to BRB breakdown in DR. Thus, inhibition of ADA2 by miR-146b-3p is a potential therapeutic strategy to preserve retinal endothelial cell barrier function in DR.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.