Trained, personnel in this core facility will utilize sophisticated state of the art technologies to address and quantitate the physiological significance of the studies proposed in the program project. These important tools include: 1) measurement of force development, 2) determination of monolayer electrical resistance (current impedance), 3) endothelial cell imaging and gap assessment, and 4) quantitation of monolayer permeability. Measurement of force development provides direct quantitation of active tension resulting from activation of the endothelial cell contractile apparatus. Electrical resistance quantitation provides rapid assessment of the opening of conductive gaps (between the borders of adjacent endothelial cells) and focal adhesions (between endothelium and the extracellular matrix. Video imaging of live cells and histologic and confocal imaging of fixed and immunofluorescent-stained cells provides important additional information about the time course, nature, number, and size of paracellular gaps and the overall changes in cell shape. Immunofluorescent imaging furnishes information on the location and organization of key regulatory, contractile, and tethering proteins. Interference reflection microscopy permits quantitation of the changes in focal adhesions between endothelial cells and the matrix. Permeability assessments provides a direct physiological correlate of the critical barrier function that the endothelial cells provide against loss of vascular proteins by quantitation of the rate of albumin transfer across the monolayer grown on a semi-permeable membrane support. Additional services offered by the core are microinjection and measurement of [Ca2+]. Microinjection provides a direct means of altering the intracellular biochemical environment with agents that are not permeable to the plasma membrane, but which are likely to have profound effects on control of contractile and tethering protein assemblies. Ca2+ measurement (based on the fluorescent dye ration method) yields rapid quantitative assessment of one key intracellular signal that controls endothelial barrier function. The tight linkage of these complementary physiologic parameters with the biochemical and molecular results from the various projects will allow assessment of the relative contribution of altered tethering and centripetal forces in producing endothelial cell barrier dysfunction.
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