In order to establish a functional endothelium for tissue engineered arteries, we developed a novel co-culture model in which human endothelial cells (EC) form a confluent monolayer on quiescent smooth muscle cells (SMC). The co-culture model is a simplified representation of a blood vessel that permits rapid and efficient examination of a large number of experimental variables. Strong adhesion develops between EC and SMC, SMC are more differentiated in co-culture, and human co-cultures can be maintained for as long as 30 days. Integrins play an important role in endothelial force transduction. Our preliminary data suggest that EC cultured on extracellular matrix produced by SMC produce fibrillar adhesions rather than focal adhesions observed when ECs adhere to rigid substrates. Further, EC in co-culture show a reduced oxidative and inflammatory state relative to EC cultured on plastic suggesting a shift in the type of adhesion and integrins involved during co-culture may affect the function of endothelium. The shift in the type of adhesion can influence integrins involved in adhesion during co- culture and the subsequent function of ECs. To properly design tissue engineered vessels that produce appropriate EC function, it is necessary to understand the effect of EC adhesion to the matrix overlying SMC upon EC function following exposure to flow. Thus, we will test the hypotheses that (1) in co-culture, fibrillar adhesion formation and the elastic modulus of SMCs influence specific extracellular matrix proteins and integrins involved in EC adhesion;(2) fibrillar adhesions promote an anti-inflammatory and anti- thrombotic EC phenotype under static and flow conditions by regulating the level of the transcription factor KLF2, and (3) a combination of co-culture and pulsatile shear stresses lowers the permeability of endothelium by reducing the formation of actin stress fibers with fibrillar adhesions. Specific aims of the project are to: (1) identify integrins and adhesion molecules involved in fibrillar and focal adhesion formation between EC and SMC in co-culture;(2) determine the importance of SMC elasticity and fibrillar adhesions upon EC adhesion and function;(3) determine the effect of fibrillar adhesions upon the response of co-cultured EC to flow;and (4) determine the effect of long-term flow upon EC permeability in co-culture. These studies will provide important new information about EC and SMC function interactions that can influence the design of tissue-engineered blood vessels. Endothelial Cell Adhesion &Function on Smooth Muscle There is considerable need for new sources of blood vessels to repair or replace vessels damaged by atherosclerosis. Tissue engineering represents one such opportunity. The proposed research will examine the manner in which the cells that line arteries and veins (endothelial cells) attach and function on surfaces produced by smooth muscle cells. The studies will provide new insights into the manner in which these two cells of the vessel wall interact and provide a cell culture system to facilitate development of tissue- engineered arteries.