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Colin Andrew Bretz, Sara R Savage, John S Penn; Isoform-specific inhibition of NFATc1 and NFATc2 reduces VEGF- and TNFa-induced inflammation in retinal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2247. doi: https://doi.org/.
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Retinal levels of vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFa) are known to be elevated at an early disease stage in diabetic patients. In retinal endothelial cells (RMEC), VEGF and TNFa induce expression of leukocyte adhesion proteins and inflammatory cytokines that contribute to leukostasis, a complication of diabetic retinopathy (DR). The nuclear factor of activated T-cells (NFAT) family of transcription factors are also known to upregulate inflammatory proteins, and can act downstream of VEGF and TNFa in this context. We hypothesize that NFAT family isoforms play distinct roles in the expression of inflammatory proteins in response to diabetes-relevant stimuli, and the present study aimed to evaluate the roles of NFATc1 and NFATc2 under VEGF and TNFa treatment conditions.
RMEC were transfected with isoform-specific siRNA or a scrambled sequence and collected 4hrs post VEGF (25ng/ml) or TNFa (10ng/ml) treatment. Quantitative real-time RT-PCR was performed by co-amplification of VCAM1 and ICAM1 vs. β-actin. For parallel plate flow chamber (PPFC) assays, cells were grown on glass slides and transfected prior to TNFa (10ng/ml) treatment. 4hrs later, slides were placed in PPFC and peripheral blood mononuclear cells (PBMC) were flowed over the monolayer. Flow was stopped, non-adherent cells were washed off, and remaining cells were counted.
Both VEGF and TNFa stimulated increased expression of VCAM1 and ICAM1 in treated human RMEC. NFATc1 knockdown caused a 65% decrease in VEGF induced VCAM1 expression (P<0.03), while NFATc2 knockdown caused a 69% and 51% decrease in TNFa induced VCAM1 and ICAM1 expression (P<0.01 and P<0.05), respectively. TNFa stimulation of PPFC slides caused increased PBMC adhesion, which is abrogated by NFATc2 knockdown.
Here we provide evidence that two distinct NFAT isoforms play a role in the downstream signaling of VEGF and TNFa. NFATc1 modulates VEGF-induced VCAM1 expression, while NFATc2 modulates both TNFa-induced VCAM1 and ICAM1 expression. The latter effect underlies the reduction seen in adherent PBMCs when NFATc2 knockdown is combined with TNFa treatment. Together, these findings suggest that NFAT signaling may be a valuable target for DR treatment, since multiple NFAT isoforms contribute to the signaling of two distinct and pathologically relevant peptides.
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