EFFECT OF ENDOTHELIAL-CELLS AND TRANSFORMING GROWTH-FACTOR-BETA-1 ON CULTURED VASCULAR SMOOTH-MUSCLE CELL-GROWTH PATTERNS

Smooth muscle cells (SMCs) cultured alone exhibit characteristic ''hil l and valley'' macroscopic growth features. We studied smooth muscle c ells cocultured with endothelial cells and the effect of transforming growth factor beta 1 on smooth muscle cells. Bovine smooth muscle cell s were plated on 13 mu m-thick semipermeable membranes. Smooth muscle cells were cultured either alone (in Dulbecco's Modified Eagles Media/ 2.5% calf serum, four wells/group); with neutralizing anti-transformin g growth factor-beta 1 antibody (10 mu g/ml); with the protease inhibi tor aprotinin (prevents plasmin-mediated activation of transforming gr owth factor-beta 1, 200 mg/ml); or in the presence of confluent bovine endothelial cells cocultured on the opposite side of the membrane bef ore plating smooth muscle cells. After 72 hours in culture smooth musc le cell organizational growth characteristics were examined by light m icroscopy. Hill and valley formation by smooth muscle cells resulted i n areas of the membrane becoming devoid of smooth muscle cells, wherea s other areas developed multilayered densely populated smooth muscle c ells. Computed planimetry was used to measure this bare surface area t o quantitate the extent of hill and valley growth, which was compared between groups by analysis of variance. Smooth muscle cells cultured a lone demonstrated prominent hill and valley formation with a bare surf ace area of 2.64 +/- 0.51 mm(2). Smooth muscle cells exposed to transf orming growth factor-beta 1 antibody had much less hill and valley for mation (bare surface area 0.92 +/- 0.29, p < 0.01), whereas aprotinin virtually prevented hill and valley formation (bare surface area 0.0,p < 0.01). Smooth muscle cells cultured opposite endothelial cells demo nstrated an organized growth pattern without hill and valley growth (b are surface area 0.0, p < 0.01 vs smooth muscle cell alone). However, transforming growth factor-beta 1 (10 ng/ml) added to smooth muscle ce lls cocultured opposite endothelial cells resulted in hill and valley formation. These results support the hypothesis that hill and valley g rowth in cultured smooth muscle cells is due in part to transforming g rowth factor-beta 1 and can be markedly reduced by agents that reduce the activation of transforming growth factor-beta 1 (aprotinin) or dir ectly antagonize its action (blocking antibody). The presence of endot helial cells in coculture also block this hill and valley growth, pres umably by inhibiting the transforming growth factor-beta 1 effect, bec ause this was overcome by additional transforming growth factor-beta 1 .