title: miR-483 Targeting of CTGF Suppresses Endothelial-to-Mesenchymal Transition: Therapeutic Implications in Kawasaki Disease.
abstract: BACKGROUND
  Endothelial-mesenchymal transition (EndoMT) is implicated in myofibroblast-like cell-mediated damage to the coronary arterial wall in acute Kawasaki disease (KD) patients, as evidenced by positive staining for connective tissue growth factor (CTGF) and EndoMT markers in KD autopsy tissues. However, little is known about the molecular basis of EndoMT involved in KD.
  
  
  OBJECTIVE
  We investigated the microRNA (miRNA) regulation of CTGF and the consequent EndoMT in KD pathogenesis. As well, the modulation of this process by statin therapy was studied.
  
  
  RESULTS
  Sera from healthy children and KD subjects were incubated with human umbilical vein endothelial cells. Cardiovascular disease-related miRNAs, CTGF, and EndoMT markers were quantified using reverse transcriptase quantitative polymerase chain reaction, ELISA, and Western blotting. Compared with healthy controls, human umbilical vein endothelial cell incubated with sera from acute KD patients had decreased miR-483, increased CTGF, and increased EndoMT markers. Bioinformatics analysis followed by functional validation demonstrated that Krüppel-like factor 4 (KLF4) transactivates miR-483, which in turn targets the 3' untranslated region of CTGF mRNA. Overexpression of KLF4 or pre-miR-483 suppressed, whereas knockdown of KLF4 or anti-miR-483 enhanced, CTGF expression in endothelial cells in vitro and in vivo. Furthermore, atorvastatin, currently being tested in a phase I/IIa clinical trial in KD children, induced KLF4-miR-483, which suppressed CTGF and EndoMT in endothelial cells.
  
  
  CONCLUSIONS
  KD sera suppress the KLF4-miR-483 axis in endothelial cells, leading to increased expression of CTGF and induction of EndoMT. This detrimental process in the endothelium may contribute to coronary artery abnormalities in KD patients. Statin therapy may benefit acute KD patients, in part, through the restoration of KLF4-miR-483 expression.
  
  
  BACKGROUND
  URL: http://www.clinicaltrials.gov. Unique identifier: NCT01431105.
authors.affiliation: From the Cardiovascular Research Center, Key Laboratory of Environment and Genes Related to Diseases (M.H., J.Z., J.Y.-J.S.), Department of Rheumatology, First Affiliated Hospital (M.H.), Xi'an Jiaotong University Health Science Center, China; Division of Cardiology, Department of Medicine (M.H., Z.C., M.M., B.W., J.Y.-J.S.) and Department of Pediatrics (A.H.T., C.S., J.C.B.), University of California, San Diego; Rady Children's Hospital, San Diego, CA (A.H.T., J.C.B.); Division of Biochemistry and Molecular Biology, University of California, Riverside (M.M.); and Case Cardiovascular Research Institute, School of Medicine, Case Western Reserve University, Cardiovascular Research Center, Cleveland, OH (P.S., M.K.J.).
authors.forename: Ming
authors.initials: M
authors.lastname: He
authors.affiliation: From the Cardiovascular Research Center, Key Laboratory of Environment and Genes Related to Diseases (M.H., J.Z., J.Y.-J.S.), Department of Rheumatology, First Affiliated Hospital (M.H.), Xi'an Jiaotong University Health Science Center, China; Division of Cardiology, Department of Medicine (M.H., Z.C., M.M., B.W., J.Y.-J.S.) and Department of Pediatrics (A.H.T., C.S., J.C.B.), University of California, San Diego; Rady Children's Hospital, San Diego, CA (A.H.T., J.C.B.); Division of Biochemistry and Molecular Biology, University of California, Riverside (M.M.); and Case Cardiovascular Research Institute, School of Medicine, Case Western Reserve University, Cardiovascular Research Center, Cleveland, OH (P.S., M.K.J.).
authors.forename: Zhen
authors.initials: Z
authors.lastname: Chen
authors.affiliation: From the Cardiovascular Research Center, Key Laboratory of Environment and Genes Related to Diseases (M.H., J.Z., J.Y.-J.S.), Department of Rheumatology, First Affiliated Hospital (M.H.), Xi'an Jiaotong University Health Science Center, China; Division of Cardiology, Department of Medicine (M.H., Z.C., M.M., B.W., J.Y.-J.S.) and Department of Pediatrics (A.H.T., C.S., J.C.B.), University of California, San Diego; Rady Children's Hospital, San Diego, CA (A.H.T., J.C.B.); Division of Biochemistry and Molecular Biology, University of California, Riverside (M.M.); and Case Cardiovascular Research Institute, School of Medicine, Case Western Reserve University, Cardiovascular Research Center, Cleveland, OH (P.S., M.K.J.).
authors.forename: Marcy
authors.initials: M
authors.lastname: Martin
authors.affiliation: From the Cardiovascular Research Center, Key Laboratory of Environment and Genes Related to Diseases (M.H., J.Z., J.Y.-J.S.), Department of Rheumatology, First Affiliated Hospital (M.H.), Xi'an Jiaotong University Health Science Center, China; Division of Cardiology, Department of Medicine (M.H., Z.C., M.M., B.W., J.Y.-J.S.) and Department of Pediatrics (A.H.T., C.S., J.C.B.), University of California, San Diego; Rady Children's Hospital, San Diego, CA (A.H.T., J.C.B.); Division of Biochemistry and Molecular Biology, University of California, Riverside (M.M.); and Case Cardiovascular Research Institute, School of Medicine, Case Western Reserve University, Cardiovascular Research Center, Cleveland, OH (P.S., M.K.J.).
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: From the Cardiovascular Research Center, Key Laboratory of Environment and Genes Related to Diseases (M.H., J.Z., J.Y.-J.S.), Department of Rheumatology, First Affiliated Hospital (M.H.), Xi'an Jiaotong University Health Science Center, China; Division of Cardiology, Department of Medicine (M.H., Z.C., M.M., B.W., J.Y.-J.S.) and Department of Pediatrics (A.H.T., C.S., J.C.B.), University of California, San Diego; Rady Children's Hospital, San Diego, CA (A.H.T., J.C.B.); Division of Biochemistry and Molecular Biology, University of California, Riverside (M.M.); and Case Cardiovascular Research Institute, School of Medicine, Case Western Reserve University, Cardiovascular Research Center, Cleveland, OH (P.S., M.K.J.).
authors.forename: Panjamaporn
authors.initials: P
authors.lastname: Sangwung
authors.affiliation: From the Cardiovascular Research Center, Key Laboratory of Environment and Genes Related to Diseases (M.H., J.Z., J.Y.-J.S.), Department of Rheumatology, First Affiliated Hospital (M.H.), Xi'an Jiaotong University Health Science Center, China; Division of Cardiology, Department of Medicine (M.H., Z.C., M.M., B.W., J.Y.-J.S.) and Department of Pediatrics (A.H.T., C.S., J.C.B.), University of California, San Diego; Rady Children's Hospital, San Diego, CA (A.H.T., J.C.B.); Division of Biochemistry and Molecular Biology, University of California, Riverside (M.M.); and Case Cardiovascular Research Institute, School of Medicine, Case Western Reserve University, Cardiovascular Research Center, Cleveland, OH (P.S., M.K.J.).
authors.forename: Brian
authors.initials: B
authors.lastname: Woo
authors.affiliation: From the Cardiovascular Research Center, Key Laboratory of Environment and Genes Related to Diseases (M.H., J.Z., J.Y.-J.S.), Department of Rheumatology, First Affiliated Hospital (M.H.), Xi'an Jiaotong University Health Science Center, China; Division of Cardiology, Department of Medicine (M.H., Z.C., M.M., B.W., J.Y.-J.S.) and Department of Pediatrics (A.H.T., C.S., J.C.B.), University of California, San Diego; Rady Children's Hospital, San Diego, CA (A.H.T., J.C.B.); Division of Biochemistry and Molecular Biology, University of California, Riverside (M.M.); and Case Cardiovascular Research Institute, School of Medicine, Case Western Reserve University, Cardiovascular Research Center, Cleveland, OH (P.S., M.K.J.).
authors.forename: Adriana H
authors.initials: AH
authors.lastname: Tremoulet
authors.affiliation: From the Cardiovascular Research Center, Key Laboratory of Environment and Genes Related to Diseases (M.H., J.Z., J.Y.-J.S.), Department of Rheumatology, First Affiliated Hospital (M.H.), Xi'an Jiaotong University Health Science Center, China; Division of Cardiology, Department of Medicine (M.H., Z.C., M.M., B.W., J.Y.-J.S.) and Department of Pediatrics (A.H.T., C.S., J.C.B.), University of California, San Diego; Rady Children's Hospital, San Diego, CA (A.H.T., J.C.B.); Division of Biochemistry and Molecular Biology, University of California, Riverside (M.M.); and Case Cardiovascular Research Institute, School of Medicine, Case Western Reserve University, Cardiovascular Research Center, Cleveland, OH (P.S., M.K.J.).
authors.forename: Chisato
authors.initials: C
authors.lastname: Shimizu
authors.affiliation: From the Cardiovascular Research Center, Key Laboratory of Environment and Genes Related to Diseases (M.H., J.Z., J.Y.-J.S.), Department of Rheumatology, First Affiliated Hospital (M.H.), Xi'an Jiaotong University Health Science Center, China; Division of Cardiology, Department of Medicine (M.H., Z.C., M.M., B.W., J.Y.-J.S.) and Department of Pediatrics (A.H.T., C.S., J.C.B.), University of California, San Diego; Rady Children's Hospital, San Diego, CA (A.H.T., J.C.B.); Division of Biochemistry and Molecular Biology, University of California, Riverside (M.M.); and Case Cardiovascular Research Institute, School of Medicine, Case Western Reserve University, Cardiovascular Research Center, Cleveland, OH (P.S., M.K.J.).
authors.forename: Mukesh K
authors.initials: MK
authors.lastname: Jain
authors.affiliation: From the Cardiovascular Research Center, Key Laboratory of Environment and Genes Related to Diseases (M.H., J.Z., J.Y.-J.S.), Department of Rheumatology, First Affiliated Hospital (M.H.), Xi'an Jiaotong University Health Science Center, China; Division of Cardiology, Department of Medicine (M.H., Z.C., M.M., B.W., J.Y.-J.S.) and Department of Pediatrics (A.H.T., C.S., J.C.B.), University of California, San Diego; Rady Children's Hospital, San Diego, CA (A.H.T., J.C.B.); Division of Biochemistry and Molecular Biology, University of California, Riverside (M.M.); and Case Cardiovascular Research Institute, School of Medicine, Case Western Reserve University, Cardiovascular Research Center, Cleveland, OH (P.S., M.K.J.). jshyy@ucsd.edu jcburns@ucsd.edu.
authors.forename: Jane C
authors.initials: JC
authors.lastname: Burns
authors.affiliation: From the Cardiovascular Research Center, Key Laboratory of Environment and Genes Related to Diseases (M.H., J.Z., J.Y.-J.S.), Department of Rheumatology, First Affiliated Hospital (M.H.), Xi'an Jiaotong University Health Science Center, China; Division of Cardiology, Department of Medicine (M.H., Z.C., M.M., B.W., J.Y.-J.S.) and Department of Pediatrics (A.H.T., C.S., J.C.B.), University of California, San Diego; Rady Children's Hospital, San Diego, CA (A.H.T., J.C.B.); Division of Biochemistry and Molecular Biology, University of California, Riverside (M.M.); and Case Cardiovascular Research Institute, School of Medicine, Case Western Reserve University, Cardiovascular Research Center, Cleveland, OH (P.S., M.K.J.). jshyy@ucsd.edu jcburns@ucsd.edu.
authors.forename: John Y-J
authors.initials: JY
authors.lastname: Shyy
mesh_terms: D000818:Animals; D000069059:Atorvastatin / Q000008:administration & dosage*; D002417:Cattle; D002675:Child, Preschool; D055513:Connective Tissue Growth Factor / Q000096:biosynthesis* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects / Q000502:physiology*; D005260:Female; D018390:Gene Targeting / Q000379:methods*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000008:administration & dosage; D007223:Infant; D000090062:Kruppel-Like Factor 4; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D035683:MicroRNAs / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics; D009080:Mucocutaneous Lymph Node Syndrome / Q000097:blood* / Q000235:genetics / Q000628:therapy*
chemical_list: C526317:CCN2 protein, human; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; C548912:MIRN483 microRNA, human; D035683:MicroRNAs; D055513:Connective Tissue Growth Factor; D000069059:Atorvastatin
keywords: CTGF; EndoMT; Kawasaki disease; atorvastatin; endothelial dysfunction; miR-483; microRNA