Figure 3. Schematic representation of the experimental strategy we used to show that in addition to the media, the adventitia of the blood vessel also presents a vasoactive response. VSMCs and vascular fibroblasts (VFs) were isolated and amplified from human saphenous vein. After 10 to 15 days of culture in appropriate medium supplemented with sodium ascorbate to stimulate extracellular matrix (ECM) synthesis, cells formed thick living tissue sheets, comprising cells embedded in the ECM they secreted, that could be peeled off from the culture flask using fine forceps. TEVM and TEVA constructs were obtained by wrapping a tissue sheet of VSMCs or VFs around a tubular support (inside diameter = three mm) in order to obtain a four-layer thick construct of VSMCs or VFs, respectively. For the TEVMA constructs, a sheet was wrapped over the tubular support with two layers of VSMCs and two layers of VFs to obtain a construct that had the same number of layers (four) as the TEVM and the TEVA constructs. After a week of maturation, each construct was cut into 5-mm-long rings, while remaining on the tubular support. These rings were further cultured for two weeks. For vasoconstriction studies, rings of TEVM, TEVA, and TEVMA were removed from their support, rinsed in physiological salt solution (Kreb’s solution) and mounted in isolated organ baths containing Kreb’s solution maintained at 37°C and gassed with a mixture of 95% O₂, 5% CO₂ (pH 7.4). After three rinses and the return to a baseline tension, tissues were challenged with different concentrations of endothelins added in the bath. The response of the constructs is indicated. (The authors are thankful to Danielle Larouche, LOEX, for this picture.)