Purpose: Retinal fibrosis, characterized by fibroblasts, myofibroblasts, and extracellular matrix formation, occurs on the surface of the retinas following retinal neovascularization and vitreous hemorrhage. We have previously shown the contribution of neutrophils in the blood-retinal barrier alteration in an animal model of diabetic retinopathy (DR). We evaluated the hypothesis that the neutrophil signaling pathway contributes to the development of retinal fibrosis in DR.
Methods: Neutrophil extracellular traps (NETs) were prepared by treating human neutrophils with 25 mM glucose and 250ug advanced glycation endproducts. Transendothelial resistance of NETs and disulfiram (NET inhibitor) in human retinal endothelial cells was measured by electric cell-substrate Impedance Sensing (ECIS). The mRNA and protein expression of NETs, fibrosis, and endothelial mesenchymal transition (EndoMT) markers were analyzed on Streptozotocin-induced diabetic mice (3 months). NETs marker (Citrullinated Histone H3) was measured on serum samples of diabetic and nondiabetic subjects (n=10 in each group).
Results: Endothelial monolayer permeability was increased by NETs and significantly reduced by 10μM disulfiram. mRNA expression of EndoMT markers (TGFb1, MMP2, MMP9, ICAM1, VCAM1, & VE-Cad) and protein expression of NETs markers (PAD4 & Cit H3) were significantly increased in the retinas of diabetic animals compared with nondiabetic. Serum levels of Cit H3 were significantly increased in diabetic compared with nondiabetic subjects.
Conclusions: Overall, we have shown evidence that neutrophil extracellular traps triggered by neutrophil infiltration in diabetes play an essential role in EndoMT process and fibrosis. Targeting the NETs may be a potential therapeutic approach to preventing retinal fibrosis formation and traction retinal detachment.
F. Monickaraj: None. A. Das: None. J. Smoake: None.
NIH 5R01EY0286061I01BX005348-01A1
