RNA-Seq Reveals a Role for NFAT-Signaling in Human Retinal Microvascular Endothelial Cells Treated with TNFα

January RNA-Seq Reveals a Role for NFAT-Signaling in Human Retinal Microvascular Endothelial Cells Treated with TNF Sara R. Savage 0 1 2 3 Colin A. Bretz 0 1 2 3 John S. Penn 0 1 2 3 0 Funding: This work was funded by grants from the National Eye institute: R01-EY07533 (JSP) and R01- EY023639 (JSP), the National Institute of General Medical Sciences: T32-GM007628, the National Center for Advancing Translational Sciences: UL1- TR000445, and an unrestricted grant from Research to Prevent Blindness, Inc. The sequencing was performed by the Vanderbilt VANTAGE core which is funded by a grant from the National Eye Institute: P30-EY08126. The funders had no role in study 1 Data Availability Statement: All raw data files are available from the NCBI Short Read Archive database (accession number SRP047271) 2 Academic Editor: Alan Stitt, Queen's University Belfast, UNITED KINGDOM 3 1 Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America, 2 Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America, 3 Department of Ophthalmology and Visual Sciences, Vanderbilt University School of Medicine , Nashville, Tennessee , United States of America TNF has been identified as playing an important role in pathologic complications associated with diabetic retinopathy and retinal inflammation, such as retinal leukostasis. However, the transcriptional effects of TNF on retinal microvascular endothelial cells and the different signaling pathways involved are not yet fully understood. In the present study, RNA-seq was used to profile the transcriptome of human retinal microvascular endothelial cells (HRMEC) treated for 4 hours with TNF in the presence or absence of the NFAT-specific inhibitor INCA-6, in order to gain insight into the specific effects of TNF on RMEC and identify any involvement of NFAT signaling. Differential expression analysis revealed that TNF treatment significantly upregulated the expression of 579 genes when compared to vehicletreated controls, and subsequent pathway analysis revealed a TNF-induced enrichment of transcripts associated with cytokine-cytokine receptor interactions, cell adhesion molecules, and leukocyte transendothelial migration. Differential expression analysis comparing TNF-treated cells to those co-treated with INCA-6 revealed 10 genes whose expression was significantly reduced by the NFAT inhibitor, including those encoding the proteins VCAM1 and CX3CL1 and cytokines CXCL10 and CXCL11. This study identifies the transcriptional effects of TNF on HRMEC, highlighting its involvement in multiple pathways that contribute to retinal leukostasis, and identifying a previously unknown role for NFATsignaling downstream of TNF. - Diabetic retinopathy (DR) is one of the leading causes of irreversible vision loss in the US, blinding approximately 12% of diabetic patients every year.[1,2] Inflammation is an important component of DR, with a number of cytokines and adhesion proteins induced by or increased in the diabetic milieu that play significant roles in diabetes-induced retinal pathology.[3] design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Tumor necrosis factor-alpha (TNF) is one such soluble pro-inflammatory cytokine, and multiple reports have observed increased vitreous levels in patients with DR.[46] In particular, TNF is implicated as a contributing factor in the development of retinal leukostasis, with both pharmacologic blockade and genetic deletion of TNF having been shown to inhibit leukostasis in diabetic rodents.[7,8] Leukostasis is the firm adherence of myeloid-derived cells to the endothelium, and is a common pathogenic feature of DR often associated with chronic retinal inflammation. Increased numbers of adherent leukocytes are observed in the retinas of DR patients, where they co-localize with dead or injured endothelial cells.[9,10] Adherent leukocytes can further damage the retinal endothelium by secreting proteolytic enzymes and/or occluding retinal capillaries, ultimately leading to focal ischemia and apoptosis of cells associated with the capillary unit.[11,12] Focal ischemia causes the surrounding tissue to become hypoxic and increases the production of vasoactive factors that promote pathologic neovascularization, which is considered to be a defining feature of late stage DR.[3] These findings indicate an important role for TNF in the overall pathology of retinal leukostasis and progression of retinopathy; but the transcriptional effects of TNF on retinal microvascular endothelial cells (RMEC) are not completely understood. The nuclear factor of activated T-cell (NFAT) signaling pathway is one of many activated by TNF, and numerous TNF-induced inflammatory proteins are also known NFAT family gene targets, though to date no studies have identified a role for NFAT signaling in the context of TNF-treated retinal vascular endothelium.[1319] NFAT is a family of five proteins grouped for their similarity to Rel/NF-B family transcription factors. NFATc denotes the four isoforms (NFATc1, NFATc2, NFATc3, and NFATc4) regulated by the serine phosphatase calcineurin (CN).[20,21] CN activates NFATc proteins through its binding to a conserved Ca2+/CN-dependent translocation regulatory domain, and this association can be effectively disrupted using the small organic molecule Inhibitor of NFAT-calcineurin Association-6 (INCA-6), which competitively binds to the discrete NFAT binding site of CN, blocking NFAT activity without altering CN phosphatase activity.[22,23] In the present study, we investigated the transcriptional effect of TNF on human retinal microvascular endothelial cells (HRMEC), and whether NFAT signaling contributes to this response, by performing RNA-seq analysis on primary HRMEC treated with TNF in both the presence and absence of the NFAT-specific inhibitor INCA-6. These data characterize the role of TNF-induced inflammation on HRMEC and give insight into new therapeutic targets for DR. Materials and Methods RMEC cell culture Primary HRMEC (catalog #ACBRI 181) were purchased from Cell Systems (Kirkland, WA) and were cultured in flasks coated with attachment factor (Cell Signaling; Danvers, MA). Growth medium consisted of endothelial basal medium (EBM; Lonza; Walkersville, MD) supplemented with 10% FBS and endothelial cell growth supplements (EGM SingleQuots; Lonza). All cultures were incubated at 37C, in 5% CO2 and 95% relative humidity. Passage 3 cells were used for these experiments. Treatment and RNA isolation HRMEC were cultured to near confluence in 6-well dishes coated with attachment factor, before being serum starved (0.5% FBS in EBM) for 12 hrs. Cells were then treated with 1 ng/ml TNF (Sigma-Aldrich; St. Louis, MO) in the presence or absence of 1.0 M INCA-6 (Tocr (...truncated)