Introduction and Objective: Inflammation is associated with progression of diabetic retinopathy (DR). Inflammatory cytokines tumor necrosis factor alpha (TNFα), interleukin-1β (IL-1β), and interleukin-6 (IL-6) are elevated in DR and trigger retinal microvascular leakage. RMEC tight junction complexes include the transmembrane proteins occludin and ZO-1. Diabetic relevant stimuli cause changes in occludin and ZO-1 localization and content. Our goal was to compare the effects of TNFα, IL-1β, and IL-6 on ZO-1 and occludin in RMECs and on RMEC permeability.Methods: Human RMECs (HRMECs) were treated with TNFα, or IL-1β, or IL-6 + soluble IL-6 receptor, or vehicle, and used to perform Western blotting for ZO-1 and Occludin. HRMECs treated as above were immunostained for ZO-1 and occludin and imaged at 40x with a confocal microscope. Transendothelial electrical resistance (TEER) of HRMEC monolayers treated as above was measured with the electrical cell-substrate impedance sensing (ECIS) method. Wild-type C57bl6 mice received intravitreal injections of TNFα, or IL-1β, or IL6 + soluble IL-6 receptor, or vehicle and quantitative fluorescein angiography (qFA) was performed to evaluate the effect of cytokines on retinal vascular permeability.Results: TNFα and IL-6 decreased the occludin protein content of HRMECs (TNFα: p= 0.0129, n=8, IL-6: p=0.0162, n=8) without changing the ZO-1 content (TNFα: p= 0.0724, n=8, IL-6: p=0.1706, n=8). IL-1β treatment decreased the ZO-1 protein content of HRMECs (p= 0.0174, n=8) but did not change the occludin protein content (p=0.6073, n=8). ECIS experiments showed decreased barrier resistance of HRMEC monolayers treated with TNFα, IL-1β, and IL-6. All 3 inflammatory cytokines induced retinal vascular hyperpermeability in vivo on qFA.Conclusion: TNFα, IL-1β, and IL-6 differentially alter HRMEC tight junctional complex proteins ZO-1 and occludin to increase retinal microvascular permeability.
I. De la Huerta: None.
National Institutes of Health (EY032620)
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