A multilayer design of parallel‐plate flow chamber for studies of endothelial cell response to fluid shear stress

Fluid shear stress (FSS) has been shown to be an ubiquitous stimulator of mammalian cell metabolism. Although many of the intracellular signal transduction pathways have been characterized, the primary mechanoreceptor for FSS remains unknown. One hypothesis is that the cytoplasmic membrane acts as the receptor for FSS, leading to increased membrane fluidity, which in turn leads to the activation of heterotrimetric G proteins (13). 9-(Dicyanovinyl)-julolidine (DCVJ) is a fluorescent probe that integrates into the cell membrane and changes its quantum yield with the viscosity of the environment. In a parallel-plate flow chamber, confluent layers of DCVJ-labeled human endothelial cells were exposed to different levels of FSS. With increased FSS, a reduced fluorescence intensity was observed, indicating an increase of membrane fluidity. Step changes of FSS caused an approximately linear drop of fluorescence within 5 s, showing fast and almost full recovery after shear cessation. A linear dose-response relationship between shear stress and membrane fluidity changes was observed. The average fluidity increase over the entire cell monolayer was 22% at 26 dyn/cm2. This study provides evidence for a link between FSS and membrane fluidity, and suggests that the membrane is an important flow mechanosensor of the cell.

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Three Distinct Types of Morphological Responses of Cultured Vascular Endothelial Cells to Physiological Levels of Fluid Shear Stress

1st International Conference on Microchannels and Minichannels ◽

10.1115/icmm2003-1124 ◽

2003 ◽

Cited By ~ 1

Author(s):

Michiaka Masuda ◽

Keigi Fujiwara

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Random Walk ◽

Endothelial Cell ◽

Parallel Plate ◽

Fluid Shear Stress ◽

Vascular Endothelial Cells ◽

Vascular Endothelial ◽

Fluid Shear ◽

Flow Apparatus

Vascular endothelial cells are known to respond to fluid shear stress. To gain insights into the mechanism of flow response by these cells, various types of in vitro devices in which endothelial cells can be cultured under flowing culture medium have been designed. Using such a device, one can apply known levels of (usually laminar) fluid shear stress to cultured endothelial cells. We have made two types of devices: a viscometer-based cone-and-plate flow apparatus and a parallel plate chamber. The cone-and-plate apparatus is used to do biochemical analyses of flow effects on cells while the parallel plate chamber is used to observe dynamic behavior of endothelial cells under flow. We were able to maintain confluent endothelial cell cultures under flow for over a week in the parallel plate flow apparatus. Using this chamber and high resolution time-lapse video microscopy, we studied morphological changes of endothelial cells exposed to different levels of fluid shear stress. We found that endothelial cells in a confluent monolayer exhibited three types of fluid shear stress level-dependent morphological and motile responses within a narrow fluid shear stress range between 0.1–10 dyn/cm2. Endothelial cells cultured under no flow exhibited variable shapes and no preferred orientation of their long cell axes and showed a jiggling motion. When exposed to fluid shear stress levels of below 0.5 dyn/cm2, endothelial cell morphology and motility were not affected. However, when fluid shear stress levels were increased to 2–4 dyn/cm2, they became polygonal and showed increased random-walk activity. Fluid shear stress over 6 dyn/cm2 caused endothelial cells to initially become polygonal and increase their random-walk activity, but they soon became elongated and aligned in the direction of flow. As the cells elongated and aligned, they migrated in the direction of flow. The average velocity of this directed cell migration was less than that of cells moving randomly under the same flow condition at earlier times. These observations indicate that endothelial cells are able to detect and respond to a surprisingly small change in fluid shear stress. It is possible that endothelial cell physiology in vivo is also regulated by small changes in fluid shear stress and that a fluid shear stress change of a few dynes per cm2 within a certain region of an artery could trigger atherogenesis in that particular location.

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