Abstract
Background and aimsWhen endothelium is cultured in wells swirled on an orbital shaker, cells at the well centre experience putatively atherogenic flow whereas those near the edge experience putatively atheroprotective flow. Transcellular transport is decreased equally in both regions, consistent with it being reduced by a mediator released from cells in one part of the well and mixed in the swirling medium. Similar effects have been inferred for pro-inflammatory changes. Here we identify the mediator and flow characteristics stimulating its release. Methods and resultsMedium conditioned by cells swirled at the edge, but not by cells swirled at the centre or cultured under static conditions, significantly reduced transendothelial transport of a low density lipoprotein (LDL)-sized tracer and tumor necrosis factor α (TNF-α)-induced activation and translocation of nuclear factor κB (NF-κB), adhesion molecule expression and monocyte adhesion. Inhibiting transcytosis similarly decreased tracer transport. Unbiased proteomics revealed that cells from the swirled edge secreted substantially more follistatin-like 1 (FSTL1) than cells from the swirled centre or from static wells. Exogenous FSTL1 reduced transport of the LDL-sized tracer and of LDL itself, as well as TNF-α-induced adhesion molecule expression. Bone morphogenetic protein 4 (BMP4) increased transport of the LDL-sized tracer and adhesion molecule expression; FSTL1 abolished these effects. ConclusionsPutatively atheroprotective flow stimulates secretion of FSTL1 by cultured endothelial cells. FSTL1 reduces transcellular transport of LDL-sized particles and of LDL itself, and inhibits endothelial activation. If this also occurs in vivo, it may account for the atheroprotective nature of such flow.
Highlights
Properties of endothelial cells (ECs) depend on the haemodynamic wall shear stress (WSS) to which the cells are exposed
vascular cell adhesion molecule 1 (VCAM-1) expression by Human Aortic Endothelial Cells (HAECs) preconditioned with human follistatinlike 1 (FSTL1) for 24 h followed by 24 h of tumor necrosis factor α (TNF-α) treatment, still with FSTL1 present, was reduced 48.7 ± 3.9% compared to HAECs treated only with TNF-α (Fig. 5A and B; n = 4; p < 0.001)
In a previous paper [7], we showed that when fluid dynamic shear stress was applied to cultured endothelial monolayers by swirling them on an orbital shaker, their permeability to low density lipoprotein (LDL)-sized particles decreased compared to the permeability seen in static culture
Summary
Properties of endothelial cells (ECs) depend on the haemodynamic wall shear stress (WSS) to which the cells are exposed. The wave induced by the swirling medium causes low magnitude, multidirectional shear towards the centre of the well, and high magnitude, uniaxial flow towards the edge. A potential problem with the method is that cells experiencing spe cific flow characteristics in one part of the well can release mediators that become well mixed in the medium and alter cell properties at other locations, confounding the true relation between WSS and EC behav iour. This can be an advantage: by growing cells only in specific
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