Abstract
Purpose :
Tumor necrosis factor alpha convertase (TACE/ADAM17) is an important regulator of inflammatory and neuroprotective responses due to its sheddase activity towards cytokines, cytokine receptors and adhesion molecules. We have previously demonstrated that TACE/ADAM17 expression and activity are up-regulated in the diabetic rat and human retina. Here we aimed to further investigate TACE/ADAM17 implication and mode of action on paracellular permeability, a critical target of hyperglycemia and a pathogenic feature of diabetic retinopathy (DR).
Methods :
Paracellular permeability in human and bovine retinal endothelial cells (HuREC and BREC, respectively) in cultures was assessed by the electrical cell-substrate impedance sensing (ECIS) technique in response to TNF alpha (20-100 ng/ml for 24 hours) and high glucose (HG; 25mM for 48 hours, conditioned medium). Western blotting and immunohistochemistry analyses were performed to assess expression levels and immunolocalization of the tight junction protein junction adhesion molecule A (JAM-A), a direct substrate of TACE/ADAM17. Inhibition of TACE/ADAM17 was achieved using a chemical inhibitor AL-4-1A1 (1 µM) or a human anti-ADAM17 inhibitory antibody D1(A12) (0.5 µM).
Results :
HG and TNF alpha, given alone or in combination, promoted a significant decrease in paracellular permeability, as determined by the reduction of trans-endothelial electrical resistance in HuREC and BREC monolayers. Treatment of the cells with AL-4-1A1 inhibitor or anti-ADAM17 antibodies halted these effects and significantly restored barrier function. In addition, we have found that JAM-A-specific epifluorescence was decreased in cells stimulated with TNF alpha or HG, given alone or in combination. However, blockade of AL-4-1A1 activity using AL-4-1A1 inhibitor AL-4-1A1 or anti-ADAM17 antibodies restored normal JAM-A-specific epifluorescence.
Conclusions :
The sum of the obtained results directly implicates TACE/ADAM17 in TNF alpha and HG-induced paracellular permeability through a mechanism involving shedding of JAM-A from the cells surface and, potentially, redistribution of intercellular junctional proteins. These data further support a role for TACE/ADAM17 in DR pathogenesis and suggest the use of specific inhibitors of this convertase as potential treatment for diabetic macular edema and DR.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.