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      Understanding mechanobiology in cultured endothelium: A review of the orbital shaker method

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          Abstract

          A striking feature of atherosclerosis is its highly non-uniform distribution within the arterial tree. This has been attributed to variation in the haemodynamic wall shear stress (WSS) experienced by endothelial cells, but the WSS characteristics that are important and the mechanisms by which they lead to disease remain subjects of intensive investigation despite decades of research. In vivo evidence suggests that multidirectional WSS is highly atherogenic. This possibility is increasingly being studied by culturing endothelial cells in wells that are swirled on an orbital shaker. The method is simple and cost effective, has high throughput and permits chronic exposure, but interpretation of the results can be difficult because the fluid mechanics are complex; hitherto, their description has largely been restricted to the engineering literature. Here we review the findings of such studies, which indicate that putatively atherogenic flow characteristics occur at the centre of the well whilst atheroprotective ones occur towards the edge, and we describe simple mathematical methods for choosing experimental variables that avoid resonance, wave breaking and uncovering of the cells. We additionally summarise a large number of studies showing that endothelium cultured at the centre of the well expresses more pro-inflammatory and fewer homeostatic genes, has higher permeability, proliferation, apoptosis and senescence, and shows more endothelial-to-mesenchymal transition than endothelium at the edge. This simple method, when correctly interpreted, has the potential to greatly increase our understanding of the homeostatic and pathogenic mechanobiology of endothelial cells and may help identify new therapeutic targets in vascular disease.

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          Highlights

          • Endothelial cells experience pro- or anti-atherogenic flow in swirling culture wells.

          • Flow characteristics in different parts of the well can be estimated or computed.

          • Inflammatory, homeostatic and other cell properties depend on location in the well.

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          Most cited references60

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          Endothelial–mesenchymal transition in atherosclerosis

          Atherosclerosis is an inflammatory disease resulting in the hardening and thickening of the wall of arteries and the formation of plaques, which comprise immune cells, mesenchymal cells, lipids, and extracellular matrix. The source of mesenchymal cells in the atherosclerotic plaques has been under scrutiny for years. Current endothelial-lineage tracing studies indicate that the endothelium is a source for plaque-associated mesenchymal cells. Endothelial cells can acquire a mesenchymal phenotype through endothelial-mesenchymal transition (EndMT), wherein the expression of endothelial markers and functions is lost and the expression of mesenchymal cell marker and functions acquired. Furthermore, EndMT can result in delamination and migration of endothelial cell-derived mesenchymal cells into the underlying tissue. Here, we review the contribution of EndMT in vascular disease focusing on atherosclerosis and describe the major biochemical and biomechanical signalling pathways in EndMT during atherosclerosis progression. Furthermore, we address how the well-established systemic atherosclerosis risk factors might facilitate EndMT during atherosclerosis.
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            Endothelial-to-mesenchymal transition contributes to fibro-proliferative vascular disease and is modulated by fluid shear stress.

            Neointimal hyperplasia is a common feature of fibro-proliferative vascular disease and characterizes initial stages of atherosclerosis. Neointimal lesions mainly comprise smooth muscle-like cells. The presence of these lesions is related to local differences in shear stress. Neointimal cells may arise through migration and proliferation of smooth muscle cells from the media. However, a role for the endothelium as a source of smooth muscle-like cells has largely been disregarded. Here, we investigated the role of endothelial-to-mesenchymal transition (EndMT) in neointimal hyperplasia and atherogenesis, and studied its modulation by shear stress.
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              Endothelial cell sensing of flow direction.

              Atherosclerosis-prone regions of arteries are characterized by complex flow patterns where the magnitude of shear stress is low and direction rapidly changes, termed disturbed flow. How endothelial cells sense flow direction and how it impacts inflammatory effects of disturbed flow are unknown. We therefore aimed to understand how endothelial cells respond to changes in flow direction. Using a recently developed flow system capable of changing flow direction to any angle, we show that responses of aligned endothelial cells are determined by flow direction relative to their morphological and cytoskeletal axis. Activation of the atheroprotective endothelial nitric oxide synthase pathway is maximal at 180° and undetectable at 90°, whereas activation of proinflammatory nuclear factor-κB is maximal at 90° and undetectable at 180°. Similar effects were observed in randomly oriented cells in naive monolayers subjected to onset of shear. Cells aligned on micropatterned substrates subjected to oscillatory flow were also examined. In this system, parallel flow preferentially activated endothelial nitric oxide synthase and production of nitric oxide, whereas perpendicular flow preferentially activated reactive oxygen production and nuclear factor-κB. These data show that the angle between flow and the cell axis defined by their shape and cytoskeleton determines endothelial cell responses. The data also strongly suggest that the inability of cells to align in low and oscillatory flow is a key determinant of the resultant inflammatory activation.
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                Author and article information

                Contributors
                Journal
                Atherosclerosis
                Atherosclerosis
                Atherosclerosis
                Elsevier
                0021-9150
                1879-1484
                1 June 2019
                June 2019
                : 285
                : 170-177
                Affiliations
                [1]Department of Bioengineering, Imperial College London, UK
                Author notes
                []Corresponding author. Department of Bioengineering, Imperial College London, London, SW7 2AZ, UK. p.weinberg@ 123456imperial.ac.uk
                [1]

                These authors contributed equally to this work.

                Article
                S0021-9150(19)30363-6
                10.1016/j.atherosclerosis.2019.04.210
                6570700
                31096159
                7d37a41f-bbf0-4f49-b5e9-6375648a47f1
                © The Authors. Published by Elsevier Ltd.

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 21 February 2019
                : 30 March 2019
                : 4 April 2019
                Categories
                Article

                Immunology
                hemodynamics,endothelium,mechanotransduction,inflammation,signalling,model
                Immunology
                hemodynamics, endothelium, mechanotransduction, inflammation, signalling, model

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