The long term objectives are to further an understanding of the biology of endothelial cells, to demonstrate that cultured endothelia respond physiologically as their counterparts do in vivo, and that the endothelial barrier is maintained in part by autocoids. Based upon our results that vasoactive substances also modulate the endothelial cytoskeleton, the current hypotheses to be examined are that: (1) cytoskeletal stress fibers are involved with endothelial structural integrity; (2) stress fiber regulation is in part a function of the prostacyclin to thromboxane A2 (PGI2:TXa2) ratio; (3) barrier promoters such as serotonin and PGI2 stimulate actin polymerization as well as stress fiber assembly whereas stimulators of increased permeability such as histamine, leukotrienes, and TXs act conversely. A breach in the barrier could, therefore, be the consequence of extracellularly produced vasotoxic metabolites coupled with endothelial metabolic changes. Based upon the model of stress fiber-mediated permeability, we will investigate PGI2:TXA2 ratio changes as a result of endothelial cell shape. Shape changes are induced with different cultured substrates, e.g., fibronectin, collagen, epithelial matrix, or poly-HEMA. To determine endothelial monolayer permeability following treatment with barrier-mediating agents, cultures will be grown on microcarrier beads or on human acellular amnion membranes. These cultures provide a two-compartment assay to measure cell and macromolecule transit. The direct action of permeability modulators on actin polymerization will be tested by: DNAse 1 assay, ultracentrifugation, 3H-phalloidin binding to f-actin, and electron microscopy. Because biochemically diverse agonists have a similar effect on endothelial stress fiber number and surface area, the significance of second messenger regulation will be studied. Stress fiber numbers and surface area are measured with a digital image analyzer of fixed endothelial cells stained with NBD-phallicidin or rhodamine. Endothelial contractility, associated with stress fiber disassembly, may be part of the mechanism for increased permeability. Endothelium grown on a collagen matrix contracts this substrate, and this procedure can test stimulus-coupled contractile responses of endothelium to permeability altering substances. Lastly, it is anticipated a culture assay can be developed to predict whether a specific metabolite or product will enhance the endothelial barrier or increase permeability.
Showing the most recent 10 out of 64 publications
