Abstract
PurposeMacular edema (ME) is a leading cause of visual loss in a range of retinal diseases and despite the use of antivascular endothelial growth factor (anti-VEGF) agents, its successful treatment remains a major clinical challenge. Based on the indirect clinical evidence that interleukin-6 (IL-6) is a key additional candidate mediator of ME, we interrogated the effect of IL-6 on blood–retinal barrier (BRB) integrity in vitro.MethodsHuman retinal pigment epithelial cell (ARPE-19) and human retinal microvascular endothelial cell (HRMEC) monolayers were used to mimic the outer and inner BRB, respectively. Their paracellular permeability was assessed by measuring the passive permeation of 40 kDa fluorescein isothiocyanate (FITC)-dextran across confluent cells in the presence of IL-6. Transendothelial/epithelial electrical resistance (TEER) then was measured and the distribution of the tight junction protein ZO-1 was assessed by immunofluorescence using confocal microscopy.ResultsTreatment with IL-6 for 48 hours significantly increased the diffusion rate of FITC-dextran, decreased TEER, and disrupted the distribution of ZO-1 in ARPE-19 cells, which constitutively express the IL-6 transmembrane receptor, and this was reversed with IL-6R blockade. In contrast, IL-6 did not affect the paracellular permeability, TEER, or ZO-1 distribution in HRMECs.ConclusionsThese in vitro data support the hypothesis that IL-6 reversibly disrupts the integrity of ARPE-19 cells, but it does not affect HRMECs.Translational RelevanceIL-6 is a candidate therapeutic target in the treatment of outer BRB driven ME.
Highlights
Macular edema (ME) is a leading cause of visual impairment, and is associated with retinal barrier dysfunction and the consequent accumulation of extracellular fluid within the central retina.[1,2] a comprehensive understanding of its underlying pathophysiology remains elusive, vascular endothelial growth factor (VEGF) is known to have a key role in ME associated with retinal ischemia and inflammation, and intraocular injections of anti-VEGF agents are standard treatment for diabetic macula edema (DME) and retinal vein occlusion (RVO), with ranibizumab (Lucentis; Genentech, South San Francisco, CA) and aflibercept (Eylea; Regeneron Pharmaceuticals, Tarrytown, NY) being approved by the United States Food and Drug Administration and European Medicines Agency for these indications
For the inner blood retinal barrier (BRB), human retinal microvascular endothelial cells (HRMEC) cells (ACBRI 181) purchased from Cell Systems Corporation (Kirkland, WA) were cultured in MV2 growth medium (PromoCell) containing 5.5 mM glucose, 0.5 ng/mL VEGF, The paracellular permeability of ARPE-19 and HRMEC monolayers was assessed by measuring the passive permeation of fluorescein isothiocyanate (FITC)-dextran (40 kDa, Sigma-Aldrich Corp.) across confluent cells grown on Transwell filters (Costar, 12 mm diameter, 0.4 lm pore size)
To determine the effect of IL-6 on outer BRB integrity, we first assessed the effect of IL-6 on the paracellular permeability of ARPE-19 cell monolayers, which constitutively express the IL-6 transmembrane receptor,[18,19] by measuring the transcellular diffusion rate of FITC-dextran (40 kDa)
Summary
Macular edema (ME) is a leading cause of visual impairment, and is associated with retinal barrier dysfunction and the consequent accumulation of extracellular fluid within the central retina.[1,2] a comprehensive understanding of its underlying pathophysiology remains elusive, vascular endothelial growth factor (VEGF) is known to have a key role in ME associated with retinal ischemia and inflammation, and intraocular injections of anti-VEGF agents are standard treatment for diabetic macula edema (DME) and retinal vein occlusion (RVO), with ranibizumab (Lucentis; Genentech, South San Francisco, CA) and aflibercept (Eylea; Regeneron Pharmaceuticals, Tarrytown, NY) being approved by the United States Food and Drug Administration and European Medicines Agency for these indications. Switzerland) is used off-label for treating retinal diseases, including the orphan indication of uveitic ME (UME).[3,4,5] despite the visual benefits antiVEGF treatments have achieved, they fail to resolve ME in .20% of patients with DME6 and in .40% of patients with RVO and uveitis/UME,[7,8] suggesting that there are important alternative mediators of retinal barrier dysfunction Principal among these is the cytokine interleukin-6 (IL-6) as its intraocular concentration correlates with the severity of ME in a diverse range of retinal pathologies, including diabetic retinopathy, DME, RVO, and uveitis.[9,10,11,12] The impact of IL-6 on vascular permeability already has been studied in a variety of nonocular tissues and cancer.[13,14] Our group and others have reported previously that in patients with refractory UME, systemic inhibition of IL-6 signaling with the IL-6 receptor (IL-6R) monoclonal antibody tocilizumab (TCZ – Actemra; Hoffmann-La Roche Ltd, Basel, Switzerland) was beneficial at 6, 12, and 24 months,[15,16,17] in particular with regard to achieving a reduction in central retinal thickness where other therapies had failed. We designed the present laboratory study to model the inner BRB using human retinal microvascular endothelial cells (HRMEC) and the outer BRB using human retinal pigment epithelial (ARPE-19) cells, with the goal of interrogating the effects of IL-6 and IL-6R blockade on in vitro measures of BRB integrity
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