Vascular Endothelial Cell Response to Mechanical Stimuli

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As this year was the last term of this project, we examined our results obtained for the past two years form global point of view, and decided to develop some new experimental methods to overcome methodological problems in the previous studies.1. DEVELOPMENT OF A NEW TECHNIQUE TO OBSERVE ACTIN FILAMENTS IN ALIVE CELLSImmunofluorescence technique which has been utilized widely to observe intracellular actin filaments requires fixation of the cells, and it was vey difficult to observe actin filament dynamics in intact cells. To overcome this problem, we have introduced a combined gene of alpha-actinin, a bridging element of actin filaments and GFP (Green Fluorescent Protein) into vascular endothelial cells. Cells introduced such genes have autofluorescence in their alpha-actinin, and thus their actin filaments. We successfully observed actin filament dynamics in cells under static and shear-exposed conditions.2. DEVELOPMENT OF A FLOW-LOADING SYSTEM FOR ENDOTHELIAL CELLS ON ARTERIAL INTRALUMINAL SURFACEThere have been a lot of contradictions between results obtained from animal experiments and that based on cultured endothelial cells. To establish a new method compensating shortcomings of both experiments, we have developed a new flow-loading system to impose shear stress on the endothelial surface of an arterial segment, in which a piece of a cut-opened arterial segment is laid flat to expose its intraluminal surface to flow in a parallel plate chamber. We applied 1 Pa of shear stress to rabbit thoracic aortas perpendicularly to their vessel axis, and found that most of the endothelial cells changed their alignment parallel to the direction of flow in 72 hours. We concluded that this system can be widely used for the future studies on the mechanical response of vascular endothelial cells to shear stress.