title: RNA-seq identifies a role for the PPARβ/δ inverse agonist GSK0660 in the regulation of TNFα-induced cytokine signaling in retinal endothelial cells.
abstract: OBJECTIVE
  The peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is a transcription factor with roles in metabolism, angiogenesis, and inflammation. It has yet undefined roles in retinal inflammation and diabetic retinopathy (DR). We used RNA-seq to better understand the role of the antagonist and inverse agonist of PPARβ/δ, GSK0660, in TNFα-induced inflammation. Understanding the underlying mechanisms of vascular inflammation could lead to new treatments for DR.
  
  
  METHODS
  RNA was isolated from human retinal microvascular endothelial cells treated with a vehicle, TNFα, or TNFα plus GSK0660. RNA-seq was performed with a 50 bp single read protocol. The differential expression was determined using edgeR and gene ontology, and a pathway analysis was performed using DAVID. RNA-seq validation was performed using qRT-PCR using the primers for ANGPTL4, CCL8, NOV, CXCL10, and PDPK1.
  
  
  RESULTS
  TNFα differentially regulated 1,830 transcripts, many of which are involved in the cytokine-cytokine receptor interaction, chemokine signaling, and inflammatory response. Additionally, TNFα highly upregulated genes involved in leukocyte recruitment, including CCL5, CX3CL1, and CXCL10. GSK0660 differentially regulated 273 transcripts in TNFα-treated cells compared to TNFα alone. A pathway analysis revealed the enrichment of cytokine-cytokine receptor signaling. In particular, GSK0660 blocks the TNFα-induced upregulation of CCL8, a chemokine involved in leukocyte recruitment.
  
  
  CONCLUSIONS
  TNFα regulates several genes related to retinal leukostasis in retinal endothelial cells. GSK0660 blocks the effect of TNFα on the expressions of cytokines involved in leukocyte recruitment, including CCL8, CCL17, and CXCL10 and it may therefore block TNFα-induced retinal leukostasis.
authors.affiliation: Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN.
authors.forename: Sara R
authors.initials: SR
authors.lastname: Savage
authors.affiliation: Department of Ophthalmology and Visual Sciences, Vanderbilt University School of Medicine, Nashville, TN.
authors.forename: Gary W
authors.initials: GW
authors.lastname: McCollum
authors.affiliation: Department of Ophthalmology and Visual Sciences, Vanderbilt University School of Medicine, Nashville, TN.
authors.forename: Rong
authors.initials: R
authors.lastname: Yang
authors.affiliation: Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN ; Department of Ophthalmology and Visual Sciences, Vanderbilt University School of Medicine, Nashville, TN.
authors.forename: John S
authors.initials: JS
authors.lastname: Penn
mesh_terms: D002478:Cells, Cultured; D054412:Chemokine CCL8 / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D047494:PPAR delta / Q000819:agonists / Q000037:antagonists & inhibitors; D048068:PPAR-beta / Q000819:agonists / Q000037:antagonists & inhibitors; D012171:Retinal Vessels / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D015398:Signal Transduction / Q000187:drug effects; D013450:Sulfones / Q000494:pharmacology*; D013876:Thiophenes / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
chemical_list: C515453:CCL8 protein, human; D054412:Chemokine CCL8; D016207:Cytokines; C529769:GSK0660; D047494:PPAR delta; D048068:PPAR-beta; D013450:Sulfones; C439524:TNF protein, human; D013876:Thiophenes; D014409:Tumor Necrosis Factor-alpha