The Role of NFAT Family Transcription Factors in Diabetic Retinopathy

Abstract

Diabetic retinopathy (DR) is one of the leading causes of vision loss in working age populations. Clinically, DR is divided into early non-proliferative diabetic retinopathy (NPDR), characterized by retinal vascular inflammation, hyperpermeability, and leukostasis; and late stage proliferative diabetic retinopathy (PDR), characterized by focal ischemia and neovascularization. This work evaluates the role of NFAT family transcription factors in both NPDR and PDR pathologies. TNFα, an inflammatory cytokine elevated in the vitreous of patients with DR, is implicated in the development of retinal vascular inflammation and leukostasis in NPDR. RNA-seq analysis using human retinal microvascular endothelial cells (HRMEC) treated with TNFα and a NFAT inhibitor revealed a role for NFAT signaling in the expression of leukocyte recruitment and adhesion proteins; and inhibition in both in vitro and in vivo models of leukocyte adhesion confirmed a clear role for NFAT signaling in TNFα-induced retinal leukostasis. Subsequent studies using isoform-specific siRNA evaluated the role of individual NFAT isoforms in this context, and identified distinct and sometimes counteractive roles for each. Specifically, NFATc2 and NFATc4 were found to regulate TNFα-induced leukocyte adhesion proteins and chemoattractants respectively, and knockdown of either isoform reduced leukocyte adhesion to HRMEC. VEGF is another important signaling peptide in the development of DR pathology, and has been identified as an inducer of NFAT signaling in certain cell types, though whether this also occurred in HRMEC or retinal vasculature was unclear. In vitro analysis showed that VEGF stimulates NFATc1 nuclear translocation in HRMEC, and that NFAT inhibition blocks proliferation and tube formation. Subsequent in vivo studies using the oxygen-induced retinopathy model showed that treatment with NFAT inhibitors significantly reduced retinal neovascularization. Taken together, these studies indicate that NFAT signaling contributes to both NPDR and PDR pathology, and highlight the unique roles individual NFAT isoforms play in responding to disease-relevant stimuli. Targeting individual NFAT isoforms may prove to be a valuable and effective way to target particular aspects of DR pathology, and further investigation of these transcription factors will help develop a more complete understanding of the molecular mechanisms that underlie DR.