Multidirectional flow and LRG1 in endothelial cells: potential atheroprotective role

Haemodynamic wall shear stress affects the function of arterial endothelial cells (ECs). Low magnitude, oscillatory and multidirectional shear have all been postulated to stimulate endothelial activation, whereas high magnitude and uniaxial shear are thought to promote endothelial homeostasis. The effects of shear interact with the effects of pro-inflammatory cytokines; they are mediated by complex signal transduction pathways and together may account for the patchy nature of atherosclerosis. The swirling-well system was used to investigate shear-induced endothelial activation. The method involves culturing ECs in standard multi-well plates on the platform of an orbital shaker to induce complex flow profiles. Computational fluid dynamic (CFD) simulation revealed that the swirling medium produces low magnitude multidirectional flow (LMMF) in the centre of the well and high magnitude uniaxial flow (HMUF) at the edge. A disadvantage of the method is that sheared ECs may release soluble mediators that become mixed in the swirling medium, and corrupt of apparent relations between shear and ECs properties. This drawback was resolved using a novel coating method that restricts cell growth to specific regions of the well. This modification permitted the demonstration that ECs do indeed release anti-inflammatory soluble mediators under HMMF. Leucine-rich α-2-glycoprotein 1 (LRG1) is a pro-angiogenic protein intimately linked with inflammation. Its role in endothelial activation was investigated. ECs activated by TNF-α treatment and LMMF showed higher LRG1 expression. The protein suppressed endothelial NF-κB signalling, EC adhesion molecules expression, and monocyte recruitment. Mechanistically, LRG1 caused TNFR1 shedding via the ALK5-SMAD2 pathway and the activation of ADAM10. LRG1 was highly expressed in ECs of stenotic arteries; it was found at high concentrations in the serum of critical limb ischemia patients and correlated with sTNFR1 concentrations. These data are consistent with a novel role for LRG1 in endothelial activation and a significant influence on atherogenesis.Open Acces