Cytokine stimulated vascular cell adhesion molecule-1 (VCAM-1) ectodomain release is regulated by TIMP-3

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-I 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 alpha (TNF alpha), interleukin-1 beta (IL-1 beta), or the phorbol ester PMA. Protease expression was inhibited (knocked down) with siRNA and validated using real-time PCR. Results: Proinflammatory cytokines IL-1 beta and TNF alpha 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-l 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 (c) 2005 European Society of Cardiology. Published by Elsevier B.V.. All rights reserved.