Copyright © 2005, European Society of Cardiology
Cytokine stimulated vascular cell adhesion molecule-1 (VCAM-1) ectodomain release is regulated by TIMP-3
aSchool of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK
bCardiovascular Medicine Unit, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London, W12 ONN, UK
cOntario Cancer Institute, University Health Network, Toronto, Canada
dDepartment of Biology, University of York, York, Y010 5YW, UK
eDept. of Oncology, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Box 139, Hills Road, Cambridge CB2 2XY, UK
* Corresponding author. Tel.: +44 1603 593816; fax: +44 1603 592250. Email address: j.gavrilovic{at}uea.ac.uk
Objectives: Vascular cell adhesion molecule-1 (VCAM-1) is a cell surface adhesion molecule involved in the recruitment of leukocytes to endothelial cells on arterial walls during the pathogenesis of atherosclerosis. The soluble ectodomain of VCAM-1 (sVCAM-1) is proteolytically released from the cell surface into the circulation, a process which is up-regulated in patients with cardiovascular or inflammatory disease. Here we investigate mechanisms involved in sVCAM-1 generation in response to cytokine stimulation.
Methods: VCAM-1 ectodomain release into the conditioned media of MCEC-1 murine endothelial cells and cells grown from primary aortic explants from timp3–/– mice and wild-type littermates was measured by sandwich ELISA and Western blot after stimulation with tumor necrosis factor 
(TNF), interleukin-1β (IL-1β), or the phorbol ester PMA. Protease expression was inhibited (knocked down) with siRNA and validated using real-time PCR.
Results: Proinflammatory cytokines IL-1β and TNF up-regulated VCAM-1 ectodomain release from the MCEC-1 cells, and this was dependant on p38 and mitogen-activated protein kinases (MAP kinases) and inhibited by the matrix metalloproteinase (MMP) inhibitor BB94 and tissue inhibitor of metalloproteinase (TIMP)-3, but not TIMP-1 or TIMP-2. Timp-3–/– cells exhibited greater VCAM-1 ectodomain release following cytokine stimulation than TIMP-3-expressing cells. Additionally, cytokine stimulation of MCEC-1 cells was shown to cause down-regulation of TIMP-3 expression. Knockdown of the metalloproteinase ADAM17, but not ADAM10 or ADAM12, gene expression reduced cytokine-stimulated VCAM-1 shedding.
Conclusions: TIMP-3 regulates the release of sVCAM-1 from cytokine-stimulated endothelial cells, which is mediated by ADAM17.
KEYWORDS Cytokines; Endothelial function; Matrix metalloproteinases
Abbreviations: ADAM, A Disintegrin and Metalloproteinase • MAPK, mitogen activated protein kinase • MEK, MAPK kinase • TIMP, tissue inhibitor of metalloproteinases • VCAM-1, vascular cell adhesion molecule-1
Time for primary review 27 days
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