Neutrophil elastase contributes to the pathological vascular permeability characteristic of diabetic retinopathy
Levels of neutrophil elastase, a serine protease secreted by neutrophils, are elevated in diabetes. The purpose of this study was to determine whether neutrophil elastase (NE) contributes to the diabetes-induced increase in retinal vascular permeability in mice with streptozotocin-induced diabetes, and, if so, to investigate the potential role of IL-17 in this process.
In vivo, diabetes was induced in neutrophil elastase-deficient (Elane−/−), Il-17a−/− and wild-type mice. After 8 months of diabetes, Elane−/− mice and wild-type age-matched control mice were injected with FITC-BSA. Fluorescence microscopy was used to assess leakage of FITC-BSA from the retinal vasculature into the neural retina. The level of NE in Il-17a−/− diabetic retina and sera were determined by ELISA. In vitro, the effect of NE on the permeability and viability of human retinal endothelial cells and the expression of junction proteins and adhesion molecules were studied.
Eight months of diabetes resulted in increased retinal vascular permeability and levels of NE in retina and plasma of wild-type animals. All of these abnormalities were significantly inhibited in mice lacking the elastase. The diabetes-induced increase in NE was inhibited in mice lacking IL-17. In vitro, NE increased retinal endothelial cell permeability, which was partially inhibited by a myeloid differentiation primary response 88 (MyD88) inhibitor, NF-κB inhibitor, and protease-activated receptor (PAR)2 inhibitor. NE degraded vascular endothelial-cadherin (VE-cadherin) in a concentration-dependent manner.
IL-17 regulates NE expression in diabetes. NE contributes to vascular leakage in diabetic retinopathy, partially through activation of MyD88, NF-κB and PAR2 and degradation of VE-cadherin.
KeywordsDiabetic retinopathy Elane IL-17 Neutrophil elastase Vascular permeability
Human retinal endothelial cell
Intercellular adhesion molecule 1
Inner nuclear layer
Inner plexiform layer
Myeloid differentiation primary response 88
Outer nuclear layer
Outer plexiform layer
Spectral-domain optical coherence tomography
Toll-like receptor 4
Vascular endothelial cadherin
Zona occludens 1
The authors thank D. A. Antonetti and X. Liu (University of Michigan, Ann Arbor, MI, USA) for the retinal whole mount immunostaining. Chieh Allen Lee, Katie Franke and Heather Butler (Case Western Reserve University, Cleveland, OH, USA) who maintained the mouse colonies. Dawn Smith (Case Western Reserve University) who maintained the hRECs.
HL performed molecular analyses and wrote the manuscript. EML analysed data and wrote the manuscript. AS and PRT acquired data, were involved in the analysis and interpretation of data and reviewed the manuscript. SIL performed ELISA, was involved in the analysis and interpretation of data and edited the manuscript. TSK designed experiments, acquired data and reviewed/edited the manuscript. All the authors approved the final version of the manuscript to be published. TSK is the guarantor of this work.
This work was supported by NIH grants RO1 EY022938 and R24 EY024864 (to TSK), and grants BX003604 (to TSK) and BX003403 (to PRT) from the Department of Veterans Affairs, and core grant P30 EY011373 to Case Western Reserve University. 81900884 (to HL) from The First Affiliated Hospital of Dalian Medical University. This research received no specific grant from any funding agency in commercial or not-for-profit sectors.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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