title: Green fluorescent protein (GFP) color reporter gene visualizes parvovirus B19 non-structural segment 1 (NS1) transfected endothelial modification.
abstract: BACKGROUND
  Human Parvovirus B19 (PVB19) has been associated with myocarditis putative due to endothelial infection. Whether PVB19 infects endothelial cells and causes a modification of endothelial function and inflammation and, thus, disturbance of microcirculation has not been elucidated and could not be visualized so far.
  
  
  RESULTS
  To examine the PVB19-induced endothelial modification, we used green fluorescent protein (GFP) color reporter gene in the non-structural segment 1 (NS1) of PVB19. NS1-GFP-PVB19 or GFP plasmid as control were transfected in an endothelial-like cell line (ECV304). The endothelial surface expression of intercellular-adhesion molecule-1 (CD54/ICAM-1) and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) were evaluated by flow cytometry after NS-1-GFP or control-GFP transfection. To evaluate platelet adhesion on NS-1 transfected ECs, we performed a dynamic adhesion assay (flow chamber). NS-1 transfection causes endothelial activation and enhanced expression of ICAM-1 (CD54: mean ± standard deviation: NS1-GFP vs. control-GFP: 85.3 ± 11.2 vs. 61.6 ± 8.1; P<0.05) and induces endothelial expression of EMMPRIN/CD147 (CD147: mean ± SEM: NS1-GFP vs. control-GFP: 114 ± 15.3 vs. 80 ± 0.91; P<0.05) compared to control-GFP transfected cells. Dynamic adhesion assays showed that adhesion of platelets is significantly enhanced on NS1 transfected ECs when compared to control-GFP (P<0.05). The transfection of ECs was verified simultaneously through flow cytometry, immunofluorescence microscopy and polymerase chain reaction (PCR) analysis.
  
  
  CONCLUSIONS
  GFP color reporter gene shows transfection of ECs and may help to visualize NS1-PVB19 induced endothelial activation and platelet adhesion as well as an enhanced monocyte adhesion directly, providing in vitro evidence of possible microcirculatory dysfunction in PVB19-induced myocarditis and, thus, myocardial tissue damage.
authors.affiliation: Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard-Karls-Universität Tübingen, Tübingen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Wurster
authors.forename: Catharina
authors.initials: C
authors.lastname: Pölzelbauer
authors.forename: Tanja
authors.initials: T
authors.lastname: Schönberger
authors.forename: Angela
authors.initials: A
authors.lastname: Paul
authors.forename: Peter
authors.initials: P
authors.lastname: Seizer
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Stellos
authors.forename: Andreas
authors.initials: A
authors.lastname: Schuster
authors.forename: Rene M
authors.initials: RM
authors.lastname: Botnar
authors.forename: Meinrad
authors.initials: M
authors.lastname: Gawaz
authors.forename: Boris
authors.initials: B
authors.lastname: Bigalke
mesh_terms: D001483:Base Sequence; D051926:Basigin / Q000378:metabolism; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D042783:Endothelial Cells / Q000378:metabolism / Q000821:virology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D055806:Microvessels / Q000821:virology; D009205:Myocarditis / Q000821:virology; D010322:Parvoviridae Infections / Q000821:virology; D016732:Parvovirus B19, Human / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D010973:Platelet Adhesiveness; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: C497886:BSG protein, human; D004279:DNA, Viral; C573420:ICAM1 protein, human; C106202:NS1 protein, parvovirus; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D018799:Intercellular Adhesion Molecule-1; D051926:Basigin; D049452:Green Fluorescent Proteins