title: Microfilaments facilitate TLR4-mediated ICAM-1 expression in human aortic valve interstitial cells.
abstract: BACKGROUND
  Inflammation is involved in the pathogenesis of calcific aortic valve stenosis, and aortic valve interstitial cells (AVICs) play an important role in valve disease and remodeling. We have previously shown that human AVICs express functional toll-like receptor 4 (TLR4), and express increased levels of ICAM-1 in response to stimulation with bacterial lipopolysaccharide (LPS). This study examines (1) the role of TLR4 in regulating ICAM-1 expression in human AVICs, (2) the interaction of ICAM-1 with microfilaments, and (3) the influence of microfilament integrity on ICAM-1 expression.
  
  
  METHODS
  AVICs isolated from explanted human hearts were stimulated with LPS (0.2 μg/mL) to induce ICAM-1 expression, as shown by Western blot. TLR4 activity was influenced with a neutralizing antibody or by gene knockdown with siRNA. Flow cytometry and immunofluorescence were used to characterize membrane localization of ICAM-1. Immunoprecipitation and immunofluorescence were employed to show colocalization of ICAM-1 with microfilaments. Microfilament function was influenced with an actin polymerization inhibitor as well as siRNA.
  
  
  RESULTS
  Cellular ICAM-1 levels increased 10-fold after 24 h of LPS stimulation, and this increase was significantly attenuated by prior treatment with TLR4-neutralizing antibody or TLR4 siRNA. ICAM-1 exhibited a filamentous distribution pattern after 4 h LPS stimulation, which was associated with increased total and cell-surface ICAM-1 expression. ICAM-1 colocalized and coprecipitated with the microfilament proteins F-actin and α-actinin 1. Pretreatment with the actin polymerization inhibitor latrunculin B and silencing α-actinin 1 with siRNA reduced LPS-induced total and cell-surface ICAM-1 expression.
  
  
  CONCLUSIONS
  This study demonstrates that (1) LPS-stimulated ICAM-1 expression in human AVICs is mediated by TLR4, (2) ICAM-1 interacts with microfilaments soon after LPS stimulation, and (3) microfilament disruption reduces ICAM-1 expression. These results suggest that the interaction between ICAM-1 and microfilaments facilitates LPS-induced ICAM-1 expression in human AVICs.
authors.affiliation: Department of Surgery, Division of Cardiothoracic Surgery, University of Colorado-Denver, Aurora, Colorado 80045, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Song
authors.forename: David A
authors.initials: DA
authors.lastname: Fullerton
authors.forename: David
authors.initials: D
authors.lastname: Mauchley
authors.forename: Xin
authors.initials: X
authors.lastname: Su
authors.forename: Lihua
authors.initials: L
authors.lastname: Ao
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Yang
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Cleveland
authors.forename: Xianzhong
authors.initials: X
authors.lastname: Meng
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000473:pathology; D000185:Actinin / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D001021:Aortic Valve / Q000378:metabolism* / Q000473:pathology; D001024:Aortic Valve Stenosis / Q000378:metabolism* / Q000473:pathology; D002114:Calcinosis / Q000378:metabolism / Q000473:pathology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D009205:Myocarditis / Q000378:metabolism / Q000473:pathology; D058105:Polymerization / Q000187:drug effects; D034741:RNA, Small Interfering; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: C526905:ACTN1 protein, human; D000199:Actins; D057134:Antibodies, Neutralizing; D008070:Lipopolysaccharides; D034741:RNA, Small Interfering; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D000185:Actinin; D018799:Intercellular Adhesion Molecule-1