title: Gene transfer of nitric oxide synthase: effects on endothelial biology.
abstract: OBJECTIVE
  The purpose of the study was to investigate the role of nitric oxide (NO) in monocyte-endothelial interaction by augmenting NO release via transfection of human endothelial cells (ECs) with EC NO synthase (eNOS) DNA.
  
  
  BACKGROUND
  Enhancement of NO synthesis by L-arginine or shear stress reduces endothelial adhesiveness for monocytes and inhibits atherogenesis. To elucidate further the underlying mechanism, we augmented NO synthase expression by transfection of human EC.
  
  
  METHODS
  Liposome-mediated transfection of EC was performed with a plasmid construct containing the gene encoding eNOS. Expression of eNOS was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Endothelial cells were exposed to human monocytoid cells, and adherent cells were quantitated using a computer-assisted program. Nitric oxide was measured by chemiluminescence.
  
  
  RESULTS
  The NO levels were not different in EC that were either not transfected, transfected with beta-gal or liposomes only. The nitric oxide synthase (NOS) transfection increased NO release by +60% (n = 6), which increased further when EC were stimulated by shear stress (24 h) by +137% (n = 5) as compared with untransfected, unstimulated EC (both p < 0.05). The RT-PCR revealed diminished monocyte chemotactic protein-1 (MCP-1) expression in eNOS transfected EC. There was an inverse relation between NO levels and monocyte binding (r = -0.5669, p < 0.002). Stimulation of EC with tumor necrosis factor-alpha (TNF-alpha; 250 U/ml) led to a decrease in NO synthesis, and an increase in monocyte binding. Cells transfected with NOS were resistant to both effects of TNF-alpha.
  
  
  CONCLUSIONS
  Endothelial cells transfected with eNOS synthesize an increased amount of NO; this is associated with diminished MCP-1 expression and monocyte-endothelial binding. The reduction in monocyte-endothelial binding persists even after cytokine stimulation.
authors.affiliation: Division of Cardiovascular Medicine, Stanford University, California, USA.
authors.forename: J
authors.initials: J
authors.lastname: Niebauer
authors.forename: J
authors.initials: J
authors.lastname: Dulak
authors.forename: J R
authors.initials: JR
authors.lastname: Chan
authors.forename: P S
authors.initials: PS
authors.lastname: Tsao
authors.forename: J P
authors.initials: JP
authors.lastname: Cooke
mesh_terms: D002448:Cell Adhesion / Q000235:genetics; D002460:Cell Line; D018932:Chemokine CCL2 / Q000378:metabolism; D004730:Endothelium, Vascular / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology; D018014:Gene Transfer Techniques; D006801:Humans; D008081:Liposomes; D009000:Monocytes / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D014162:Transfection
chemical_list: D018932:Chemokine CCL2; D008081:Liposomes; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase