Abstract 208: Laminar Shear Stress Decreases Endothelial Microparticles Release by a Nitric Oxide- and ABCA1-Dependent Mechanism

Abstract

Microparticles (MP) are shed-membrane sub-micron vesicles released upon cell activation or apoptosis. Their formation is associated with loss of cell membrane asymmetry and exposure of phosphatidylserine (PS), resulting from increases in floppase activities such as ABCA1. Plasma endothelial MP (EMP) are surrogate markers of endothelial dysfunction and their levels increase in patients with cardiovascular diseases. However, the mechanisms governing EMP release in vivo remain largely unknown. As nitric oxide (NO) is a key regulator of the vasoprotective effect of laminar shear stress (SS) on the endothelium, we tested the hypothesis that endothelial NO regulates EMP release under different SS profiles. Shed-annexinV+ EMPs were analyzed by flow cytometry in the medium of confluent HUVECs (passage 1-3) subjected to either high- (15 dyne/cm2; atheroprotective) or low- (1.5 dyne/cm2; atheroprone) SS conditions for 24 hrs. HUVECs exposed to high-SS released 3-fold less EMPs compared to low-SS conditions (326±37 vs. 130±23AnnV+MPs/μL, p<0.001). High-SS EMPs also externalized less PS, as estimated by the mean fluorescence of FITC-AnnV labelling (15.6±6.1 vs. 24.2±9.4 AU, p<0.001). Exposure to L-NAME (10-5M), but not D-NAME, increased high-SS-induced EMP release by over 3-fold (p<0.001) and the NO donor SNAP (10-5M) significantly decreased low-SS-induced EMP formation by 50% (p=0.05). In addition, L-NAME increased the mean fluorescence of EMP labelling under high SS (p<0.05), whereas SNAP decreased it under low SS (p<0.05). We then evaluated ABCA1 expression under these conditions. high-SS significantly decreased the expression of ABCA1 both at the mRNA and protein level by 2- and 20-fold, respectively (p<0.05 vs low SS). L-NAME augmented ABCA1 mRNA level in cells exposed to high SS (p<0.05), whereas SNAP significantly decreased ABCA1 mRNA level under low SS conditions (p<0.05). Finally, the LXRα agonist TO90131 (0.1mM) not only increased endothelial ABCA1 expression by 20-fold, but also augmented MP level under high SS conditions. Altogether, these results demonstrate that atheroprotective shear stress limits the release of procoagulant endothelial MP through a NO-dependent regulation of endothelial ABCA1 expression.