Effects of multidirectional flow on cultured endothelium

Endothelial permeability to circulating macromolecules and patterns of haemodynamic wall shear stress both vary from site to site within the arterial system. Influences of local blood flow on macromolecule transport across the endothelium may account for the patchy nature of atherosclerosis, but the routes for such transport and their modification by flow are highly controversial. The current consensus attributes the site specific formation of atherosclerotic lesions within the vasculature to low and oscillatory hemodynamic wall shear stress. However, effects of multidirectional shear stress, which occur in lesion prone regions, are not fully understood. An orbital shaker was utilised to impose chronic multidirectional shear stresses on cultured porcine aortic endothelial cells grown in multi-well plates. The orbital motion induced a wave of culture medium which rotated around the well. The shear stresses experienced by the endothelial cells at each location on the bottom of the well and at each time during the orbit were obtained using numerical methods. Spatially-resolved measurements of permeability were made by using molecular recognition between the substrate underneath the cells and different sized tracers initially placed above them. The tracer bound to the substrate once it was transported through the monolayer, and was detected by confocal microscopy. Numerical methods revealed cells at the centre of the well experienced multidirectional shear stress whereas cells at the edge were subjected to uniaxial shear stress. The transport routes of macromolecules through the monolayer were dependent on size of the macromolecules and their modification by different shear stress characteristics also varied. Chronic multidirectional shear stress appears to be proatherogenic, priming endothelial cell for inflammation and affecting the barrier function of the monolayer. Combining spatially-resolved measurements of permeability with the orbital shaker model to study the effects of chronic multidirectional flow will be a useful model for further in vitro studies.Open Acces

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

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

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.National Medical Research Council (NMRC)Published versionThe authors gratefully acknowledge a British Heart Foundation project grant (to PDW), a National Medical Research Council Singapore TAAP and DYNAMO Grant (to XW, NMRC/OFLCG/004/2018, NMRC/OFLCG/001/2017), an A*STAR Graduate Scholarship (to KTP), and a British Heart Foundation Center of Research Excellence studentship (to MA)

Leucine-Rich ?-2-Glycoprotein 1 Suppresses Endothelial Cell Activation Through ADAM10-Mediated Shedding of TNF-? Receptor

10.3389/fcell.2021.706143Frontiers in Cell and Developmental Biology970614