Abstract
AMP-activated protein kinase (AMPK) is an evolutionary conserved energy-sensing enzyme that regulates cell metabolism. Emerging evidence indicates that AMPK also plays an important role in modulating endothelial cell function. In the present study, we investigated whether AMPK modulates endothelial cell growth. Treatment of cultured human umbilical vein endothelial cells with the AMPK activators 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), 6,7-dihydro-4-hydroxy-3-(2′-hydroxy[1,1′-biphenyl]-4-yl)-6-oxo-thieno[2,3-b]pyridine-5-carbonitrile (A-769662), or metformin inhibited cell proliferation and DNA synthesis. The antiproliferative action of AICAR was largely prevented by the adenosine kinase inhibitor 5′-iodotubercidin and mimicked by infecting endothelial cells with an adenovirus expressing constitutively active AMPK. In contrast, pharmacological blockade of endothelial nitric oxide synthase or heme oxygenase-1 activity failed to reverse the inhibition of endothelial cell growth by AICAR. Flow cytometry experiments revealed that pharmacological activation of AMPK arrested endothelial cells in the G0/G1 phase of the cell cycle, and this was associated with increases in p53 phosphorylation and p53, p21, and p27 protein expression and decreases in cyclin A protein expression and retinoblastoma protein phosphorylation. In addition, silencing p21 and p27 expression partially restored the mitogenic response of AMPK-activated cells. Finally, activation of AMPK by AICAR blocked the migration of endothelial cells after scrape injury and stimulated tube formation by endothelial cells plated onto Matrigel-coated plates. In conclusion, these studies demonstrate that AMPK activation inhibits endothelial cell proliferation by elevating p21 and p27 expression. In addition, they show that AMPK regulates endothelial cell migration and differentiation and identify AMPK as an attractive therapeutic target in treating diseases associated with aberrant endothelial cell growth.
Footnotes
This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants R01-HL59976, R01-HL74966]. B.Y. was supported by a University of Missouri Summer Research Internship in Cell and Molecular Biology.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS:
- AMPK
- AMP-activated protein kinase
- AdAMPK-CA
- adenovirus expressing a constitutively active AMPK mutant
- AICAR
- 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside
- ACC
- acetyl-CoA carboxylate
- eNOS
- endothelial nitric oxide synthase
- HO
- heme oxygenase
- HUVEC
- human umbilical vein endothelial cell
- HAEC
- human aortic endothelial cell
- AdGFP
- adenovirus expressing green fluorescent protein
- Iodo
- 5-iodobercidin
- PBS
- phosphate-buffered saline
- siRNA
- small interfering RNA
- l-NMA
- NG-monomethyl-l-arginine
- SnPP
- tin protoporphyrin-IX
- pRb
- phospho-retinoblastoma protein
- NT
- nontargeting
- A-769662
- 6,7-dihydro-4-hydroxy-3-(2′-hydroxy[1,1′-biphenyl]-4-yl)-6-oxo-thieno[2,3-b]pyridine-5-carbonitrile.
- Received March 26, 2012.
- Accepted June 12, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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