High-density lipoprotein increases the abundance of eNOS protein in human vascular endothelial cells by increasing its half-life.
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.
Rämet
Maria E
ME
Molecular Cardiology Research Institute and Department of Medicine, New England Medical Center Hospitals, Inc., Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Rämet
Mika
M
Lu
Qing
Q
Nickerson
Moriah
M
Savolainen
Markku J
MJ
Malzone
Amy
A
Karas
Richard H
RH
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
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