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Meredith J Giblin, Megan E Capozzi, Gary W. McCollum, John S Penn; NFAT-dependency of IL-1β-induced diabetes-relevant behaviors in human retinal microvascular endothelial cells and Müller cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4044. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Early diabetic retinopathy (DR) involves chronic low-grade inflammation, characterized by elevated vitreous cytokines, such as IL-1β. This inflammation promotes many pathologic consequences, including the hallmark vascuar changes for which DR is best known. Nuclear factor of activated T cells (NFAT) is involved in the regulation of inflammatory mediators, extracellular matrix proteins, and adhesion molecules and thus may control multiple pathogenic steps early in DR. We have examined NFAT's role in IL-1β auto-amplification and explored the NFAT-dependency of endothelial cell responses to IL-1β, including monolayer permeability and expression of targets involved in leukocyte adhesion and basement membrane thickening.
For IL-1β production, human Müller cells (HMC) were treated with 50pg/mL IL-1β, Inhibitor of NFAT-Calcineurin Association-6 (INCA, 2.5μM) and proper vehicles for 8 hrs before collection for qRT-PCR. For ICAM expression, human retinal microvascular endothelial cells (HRMEC) were treated with 1ng/mL IL-1β and 2.5μM INCA for 2 hrs. For collagen IV expression, HRMEC were treated with 10ng/mL IL-1β and 1μM INCA for 48 hrs. In permeability experiments, HRMEC were pre-treated with vehicle or 2.5μM INCA for 16 hrs before 24 hrs of exposure to 0.5ng/mL IL-1β with/out INCA. Transendothelial electrical resistance values were measured using the EVom2.
IL-1β increased HMC IL-1β expression by 82-fold (p<0.01); INCA inhibited this induction by 28% (p<0.05). HRMEC treatment with IL-1β caused a 328-fold induction of ICAM expression (p<0.01); INCA inhibited this induction by 20% (p<0.01). HRMEC treatment with IL-1β caused a 2-fold induction of collagen IV expression (p<0.01); INCA inhibited this induction by 50% (p<0.01). Treatment with IL-1β decreased HRMEC monolayer resistance by 25% (measured vs resistance at time=0, p<0.01), which was partially rescued (44%, p<0.01) by INCA treatment.
These data demonstrate the potential of NFAT as a multi-targeted therapy for retinal inflammation secondary to diabetes. NFAT inhibition not only prevented HMC IL-1β auto-amplification, but also inhibited the pathogenic response of HRMEC to IL-1β, including the expression of adhesion proteins, excessive extracellular matrix deposition and increased permeability. Future work will investigate the therapeutic potential of in vivo NFAT inhibition.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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