title: Inhibition of cytokine signaling in human retinal endothelial cells through downregulation of sphingomyelinases by docosahexaenoic acid.
abstract: OBJECTIVE
  The authors have previously demonstrated that DHA inhibits cytokine-induced inflammation in human retinal endothelial cells (HRECs), the resident vasculature affected by diabetic retinopathy. However, the anti-inflammatory mechanism of docosahexaenoic acid (DHA) is still not well understood. Sphingolipids represent a major component of membrane microdomains, and ceramide-enriched microdomains appear to be a prerequisite for inflammatory cytokine signaling. Acid sphingomyelinase (ASMase) and neutral sphingomyelinase (NSMase) are key regulatory enzymes of sphingolipid metabolism, promoting sphingomyelin hydrolysis to proinflammatory ceramide. The authors address the hypothesis that DHA inhibits cytokine-induced inflammatory signaling in HRECs by downregulating sphingomyelinases.
  
  
  METHODS
  ASMase and NSMase activity was determined by sphingomyelinase assay in primary cultures of HRECs. The expression of ASMase, NSMase, ICAM-1, and VCAM-1 was assessed by quantitative PCR and Western blot analysis. Gene silencing of ASMase and NSMase was obtained by siRNA treatment.
  
  
  RESULTS
  Inflammatory cytokines TNFalpha and IL-1beta induced cellular adhesion molecule (CAM) expression and rapid increase in ASMase and NSMase activity in HRECs. DHA decreased basal and cytokine-induced ASMase and NSMase expression and activity and the upregulation of CAM expression. Anti-inflammatory effects of DHA on cytokine-induced CAM expression were mimicked by inhibition/gene silencing of ASMase and NSMase. The sphingomyelinase pathway rather than ceramide de novo synthesis pathway was important for inflammatory signaling in HRECs.
  
  
  CONCLUSIONS
  This study provides a novel potential mechanism for the anti-inflammatory effect of DHA in HRECs. DHA downregulates the basal and cytokine-induced ASMase and NSMase expression and activity level in HRECs, and inhibition of sphingomyelinases in endothelial cells prevents cytokine-induced inflammatory response.
authors.affiliation: Department of Physiology, Michigan State University, East Lansing, Michigan 48824, USA.
authors.forename: Madalina
authors.initials: M
authors.lastname: Opreanu
authors.forename: Todd A
authors.initials: TA
authors.lastname: Lydic
authors.forename: Gavin E
authors.initials: GE
authors.lastname: Reid
authors.forename: Kelly M
authors.initials: KM
authors.lastname: McSorley
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Esselman
authors.forename: Julia V
authors.initials: JV
authors.lastname: Busik
mesh_terms: D015153:Blotting, Western; D016207:Cytokines / Q000494:pharmacology*; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology; D020868:Gene Silencing; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000235:genetics; D012171:Retinal Vessels / Q000187:drug effects / Q000201:enzymology; D015398:Signal Transduction / Q000187:drug effects*; D021241:Spectrometry, Mass, Electrospray Ionization; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
chemical_list: D016207:Cytokines; D034741:RNA, Small Interfering; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D004281:Docosahexaenoic Acids; D013108:Sphingomyelin Phosphodiesterase