Abstract 358: Cyclic Strain-Induced Release of Thrombomodulin from Human Aortic Endothelial Cells
Introduction: Loss of thrombomodulin (TM), a cofactor in the protein C anti-coagulant pathway, through a combination of reduced TM expression and elevated TM release, is a key feature of endothelial dysfunction leading to vascular diseases. Reduction in endothelial TM levels for example is viewed as a major cause of cyclic strain-induced early vein graft failure. In this respect, our understanding of the mechanistic relationship between vessel stretch and TM expression/release in endothelial cells is very limited, with a particular shortage of in vitro models. In this study, we investigated how cyclic circumferential strain negatively regulates thrombomodulin levels in human aortic endothelial cells (HAECs) in vitro.
Methods: A Flexercell® Tension-Plus™ FX-4000T™ system (Flexcell International Corp., Hillsborough, NC) was routinely employed to subject HAECs to equibiaxial cyclic strain over a range of doses (0, 2.5 and 7.5% stretch; 60 cycles/min; cardiac waveform) and times (0-48 hours). TM expression was analyzed via Western blotting and qRT-PCR, whilst TM release was monitored by ELISA.
Results: (i) Cyclic strain of HAECs led to significant force- and time-dependent reductions in TM expression (mRNA and protein) in conjunction with elevated TM release into media; (ii) Inclusion of either GM6001 (25 μM) or apocynin (10 mM) into strain experiments to block MMP-2/9 and NADPH oxidase activities, respectively, had no effect on strain-induced TM release; (iii) Control studies demonstrated that treatment of HAECs with neither TNF-α (0, 10, 100 ng/ml) nor oxLDL (10, 50, 145.7 μg/ml) increased TM release from HAECs - however, oxLDL treatment was found to dose-dependently potentiate cyclic-strain-induced TM release.
Conclusions: Cyclic strain negatively regulates TM levels in endothelial cells in vitro. Moreover, release of TM from HAECs does not appear to involve the strain-dependent induction of either MMP-2/9 proteolytic activity or NADPH oxidase-dependent superoxide production. Finally, the cyclic strain-induced release of TM appears to be potentiated by inflammatory atherogenic conditions such as high oxLDL levels.
- © 2012 by American Heart Association, Inc.