title: Gene expression profiling of acute type A aortic dissection combined with in vitro assessment.
abstract: OBJECTIVE
  The mechanisms underlying aortic dissection remain to be fully elucidated. We aimed to identify key molecules driving dissection through gene expression profiling achieved by microarray analysis and subsequent in vitro experiments using human aortic endothelial cells (HAECs) and aortic vascular smooth muscle cells (AoSMCs).
  
  
  METHODS
  Total RNA, including microRNA (miRNA), was isolated from the intima-media layer of dissected ascending aorta obtained intraoperatively from acute type A aortic dissection (ATAAD) patients without familial thoracic aortic disease (n = 8) and that of non-dissected ascending aorta obtained from transplant donors (n = 9). Gene expression profiling was performed with mRNA and miRNA microarrays, and results were confirmed by quantitative polymerase chain reaction (qPCR). Target genes and miRNA were identified by gene ontology analysis and a literature search. To reproduce the in silico results, HAECs and AoSMCs were stimulated in vitro by upstream cytokines, and expression of target genes was assessed by qPCR.
  
  
  RESULTS
  Microarray analysis revealed 1536 genes (3.6%, 1536/42 545 probes) and 41 miRNAs (3.0%, 41/1368 probes) that were differentially expressed in the ATAAD group (versus donor group). The top 15 related pathways included regulation of inflammatory response, growth factor activity and extracellular matrix. Gene ontology analysis identified JAK2 (regulation of inflammatory response), PDGFA, TGFB1, VEGFA (growth factor activity) and TIMP3, TIMP4, SERPINE1 (extracellular matrix) as the target genes and miR-21-5p, a TIMP3 repressor, as target miRNA that interacts with the target genes. Validation qPCR confirmed the altered expression of all 7 target genes and miR-21-5p in dissected aorta specimens (all genes, P < 0.05). Ingenuity pathway analysis showed TNF-α and TGF-β to be upstream cytokines for the target genes. In vitro experiments showed these cytokines inhibit TIMP3 expression (P < 0.05) and enhance VEGFA expression (P < 0.01) in AoSMCs but not HAECs. miR-21-5p expression increases in AoSMCs under TNF-α and TGF-β stimulation (fold change: 1.36; P = 0.011).
  
  
  CONCLUSIONS
  Results of our novel approach, integrating in vitro assessment into gene expression profiling, implicated chronic inflammation characterized by MMP-TIMP dysregulation, increased VEGFA expression, and TGF-β signalling in the development of dissection. Further investigation may reveal novel diagnostic biomarkers and uncover the mechanism(s) underlying ATAAD.
authors.affiliation: Department of Cardiovascular Surgery, Saitama Medical Center, Jichi Medical University, Saitama, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Kimura
authors.affiliation: Department of Allergy and Clinical Immunology, National Research Institute for Child Health and Development, Tokyo, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Futamura
authors.affiliation: Department of Cardiothoracic Surgery, Stanford University, School of Medicine, Stanford, CA, USA.
authors.forename: Mamoru
authors.initials: M
authors.lastname: Arakawa
authors.affiliation: Department of Allergy and Clinical Immunology, National Research Institute for Child Health and Development, Tokyo, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Okada
authors.affiliation: Department of Cardiothoracic Surgery, Stanford University, School of Medicine, Stanford, CA, USA.
authors.forename: Fabian
authors.initials: F
authors.lastname: Emrich
authors.affiliation: Department of Cardiothoracic Surgery, Stanford University, School of Medicine, Stanford, CA, USA.
authors.forename: Homare
authors.initials: H
authors.lastname: Okamura
authors.affiliation: Department of Cardiothoracic Surgery, Stanford University, School of Medicine, Stanford, CA, USA.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Sato
authors.affiliation: Department of Cardiothoracic Surgery, Stanford University, School of Medicine, Stanford, CA, USA.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Shudo
authors.affiliation: Department of Cardiothoracic Surgery, Stanford University, School of Medicine, Stanford, CA, USA.
authors.forename: Tiffany K
authors.initials: TK
authors.lastname: Koyano
authors.affiliation: Department of Cardiovascular Surgery, Saitama Medical Center, Jichi Medical University, Saitama, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yamaguchi
authors.affiliation: Department of Cardiovascular Surgery, Saitama Medical Center, Jichi Medical University, Saitama, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Adachi
authors.affiliation: Department of Allergy and Clinical Immunology, National Research Institute for Child Health and Development, Tokyo, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Matsuda
authors.affiliation: Division of Cardiovascular Surgery, Department of Surgery, Jichi Medical University, Shimotsuke, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Kawahito
authors.affiliation: Department of Allergy and Clinical Immunology, National Research Institute for Child Health and Development, Tokyo, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Matsumoto
authors.affiliation: Department of Cardiothoracic Surgery, Stanford University, School of Medicine, Stanford, CA, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Fischbein
mesh_terms: D000046:Academic Medical Centers; D000704:Analysis of Variance; D000784:Aortic Dissection / Q000235:genetics* / Q000503:physiopathology; D017545:Aortic Aneurysm, Thoracic / Q000235:genetics* / Q000503:physiopathology; D015331:Cohort Studies; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D035683:MicroRNAs / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D015203:Reproducibility of Results; D018709:Statistics, Nonparametric; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics*
chemical_list: D035683:MicroRNAs; C088390:TIMP3 protein, human; D019717:Tissue Inhibitor of Metalloproteinase-3
keywords: Aortic dissection; Gene expression profiling; In vitro assessment; TIMP; miR-21-5p