The effect of environment and cell-cell interactions on endothelial cells from large vessels and microvessels will be examined in gyration mediated aggregates, either unmixed or combined with nonendothelial cell types. Both mixed and unmixed aggregates will be raised under three different in vitro conditions: normal tissue culture medium supplemented with serum (MEM- 10); MEM-10 supplemented with a known stimulator of new vessel formation; MEM-10 supplemented with either insulin or glucose to mimic the diabetic microvascular environment. Preliminary ultrastructural evidence has shown that endothelial cells cultured in this way are not only viable but also capable of sorting out and forming a network of branched interconnecting lumina. These lumenized capillary-like structures contain an abluminal extracellular matrix, junctional contacts and endothelial fenestrations. In this proposed study, the various aggregates cultured under the three conditions will be harvested for examination at 12 hours and then on days 1-7 following aggregate initiation. These aggregates will then be examined by a series of complementary techniques. Ultrastructural examination of the cell contacts, lumen formation and matrix deposition will be performed, and serial sections of the aggregates will be used to examine the process of lumen formation. Light and electron microscopic immunohistochemistry will be used to examine the qualitative differences in extracellular matrix composition among the various aggregates, and to determine if regional variations in matrix composition occur within the aggregates. To do this, antibodies against a number of extracellular matrix components (collagens I-V, laminin, fibronectin, proteoglycans) will be used to probe the aggregate microenvironment. These antibodies will also be used in enzyme-linked immunoassays to measure the actual amounts of these types of proteoglycans being synthesized will be determined and measured in extracts of the various aggregates. In addition, radiolabelling of the aggregates during culture will allow us to determine the effect of the different culture conditions and cell-cell interactions on cell proliferation. Using these various techniques, we will examine the effect of cell-cell and cell-environment interactions on endothelial cells in capillary-like three-dimensional structures using sensitive qualitative and quantitative analytical techniques.
