Endothelial cells (EC) and pericytes make frequent contact throughout the retinal microvasculature. Gap junctions have been documented between the two cell types both in vivo [Spitznas and Reale, 1975] and in vitro [Larson et al., 1987; Sweet et al., 1988]. In addition, we have shown that contact between EC and pericytes is essential for the pericyte mediated growth inhibition that we have observed in cocultures [Orlidge and D'Amore, 1987]. With these observations as evidence for the importance of EC-pericyte contacts, we now propose to characterize the molecules involved in EC-pericyte adhesion and to determine the role of the intercellular communication in maintaining normal vascular growth and function. Towards this end we will: (1) continue to develop and characterize a new system for EC-mural cell coculture. In this new co- culture chamber cells are cultured on opposite sides of a thin porous membrane. This permits intercellular contact while maintaining pure cell populations so that biochemical and molecular analyses can be conducted on the individual cell types; (2) identify the cell adhesion molecules (CAMs) on the surface of EC and pericytes that mediate their interaction. Monoclonal antibodies will be produced and screened for their ability to block EC-pericyte binding in vitro. The antibodies will subsequently be used to characterize and localize CAMs in vitro and in situ; (3) determine if EC-pericyte adhesion is regulated by exogenous factors. The expression of many CAMs is altered in response to various agents and conditions. We will undertake studies to determine if EC- pericyte interactions are similarly modulated and, using the antibodies raised above, will examine the molecular basis of the alterations; and (4) characterize the proteins which compromise gap junctions between EC and pericytes and determine their function. Monospecific antibodies and antisense mRNA against connexins, gap junction proteins, will be used to specifically inhibit the formation of gap junctions among EC and between EC and pericytes. Results from the studies outlines in this proposal will contribute to an understanding of the complex mechanisms that underlie maintenance of normal vascular growth and function and will provide insight into the events that lead to the development of retinal microangiopathy.
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