title: High-density lipoprotein increases the abundance of eNOS protein in human vascular endothelial cells by increasing its half-life.
abstract: OBJECTIVE
  Given the importance of endothelial nitric oxide synthase (eNOS) in regulating endothelium-dependent vasorelaxation, we investigated the effects of high-density lipoprotein in (HDL) on eNOS protein abundance in cultured human vascular endothelial cells.
  
  
  BACKGROUND
  Endothelial dysfunction, characterized by decreased nitric oxide production, is one of the early features in the development of atherosclerosis. We have recently shown in vivo that niacin therapy increases plasma HDL concentration and improves endothelium-dependent vasorelaxation in patients with coronary artery disease.
  
  
  METHODS
  Human vascular endothelial cells were cultured in the presence or absence of HDL or apolipoprotein (apo)A-I. The eNOS protein abundance was assessed by immunoblotting, and protein half-life was assessed by pulse-chase techniques. The eNOS messenger ribonucleic acid (mRNA) abundance was measured using real-time quantitative polymerase chain reaction.
  
  
  RESULTS
  High density lipoprotein, or apoA-I alone, increased eNOS protein abundance by 3.5 +/- 0.7 and 2.7 +/- 0.5-fold, respectively (p < 0.05 for both). However, neither HDL nor apoA-I increased eNOS mRNA abundance. It was shown that HDL increased eNOS protein half-life up to 3.3 +/- 0.2-fold (p = 0.001). Both HDL and apoA-I activated mitogen-activated protein-kinase and phosphatidylinositol 3-kinase (PI3K) Akt-pathways in human arterial endothelial cells, and inhibition of either of these pathways by specific pharmacologic inhibitors abolished the effect of HDL on eNOS.
  
  
  CONCLUSIONS
  We demonstrate that HDL activates both extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, resulting in enhanced eNOS protein stability and subsequent accumulation of eNOS protein. This posttranslational regulation represents a previously unrecognized mechanism for regulating eNOS.
authors.affiliation: Molecular Cardiology Research Institute and Department of Medicine, New England Medical Center Hospitals, Inc., Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
authors.forename: Maria E
authors.initials: ME
authors.lastname: Rämet
authors.forename: Mika
authors.initials: M
authors.lastname: Rämet
authors.forename: Qing
authors.initials: Q
authors.lastname: Lu
authors.forename: Moriah
authors.initials: M
authors.lastname: Nickerson
authors.forename: Markku J
authors.initials: MJ
authors.lastname: Savolainen
authors.forename: Amy
authors.initials: A
authors.lastname: Malzone
authors.forename: Richard H
authors.initials: RH
authors.lastname: Karas
mesh_terms: D016632:Apolipoprotein A-I / Q000737:chemistry / Q000494:pharmacology; D001161:Arteriosclerosis / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000187:drug effects*; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000187:drug effects*; D006207:Half-Life; D006801:Humans; D066298:In Vitro Techniques; D008075:Lipoproteins, HDL / Q000737:chemistry* / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects; D019001:Nitric Oxide Synthase / Q000032:analysis* / Q000737:chemistry / Q000187:drug effects*; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000187:drug effects; D044767:Ubiquitin-Protein Ligases
chemical_list: D016632:Apolipoprotein A-I; D002352:Carrier Proteins; D008075:Lipoproteins, HDL; D020928:Mitogen-Activated Protein Kinases; D019001:Nitric Oxide Synthase; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D044767:Ubiquitin-Protein Ligases; C419706:NOSIP protein, human; C494918:AKT1 protein, human