THE EFFECT OF ENDOTHELIAL-CELL COCULTURE ON SMOOTH-MUSCLE CELL-PROLIFERATION

Purpose: Smooth muscle cell (SMC) growth kinetics are often studied in culture without consideration of endothelial cell (EC) influences tha t occur in vivo. This study examined the time-dependent effect of EC o n SMC in a new type of coculture system. Methods: Bovine aortic EC and SMC were harvested from fresh specimens, grown to four passages from primary cultures, and plated on either side of a porous 13 mum thick p olyethylene terephthalate membrane. SMC were studied in coculture oppo site from confluent EC (EC/SMC). Controls included SMC cultured opposi te SMC (SMC/SMC) or SMC alone (with no cells on the opposite side of t he membrane, PHI/SMC). After cocultures were established, SMC were har vested from 1 to 4 days after release from growth arrest (n = 5 cultur es/day/group). SMC DNA and protein content and H-3-thymidine incorpora tion were measured in each group. SMC proliferation was indexed by H-3 -thymidine incorporation per cellular DNA content. Results: EC stimula ted SMC proliferation 56% more than SMC/SMC cultures and 244% more tha n SMC alone on day 1 after growth arrest (p < 0.05). This effect decre ased with time so that by day 4, EC seemed to inhibit SMC proliferatio n (49% less proliferation than SMC/SMC and 76% less than SMC alone, p < 0.05). SMC opposite EC had significantly less protein/DNA than contr ol SMC, and they retained a thin, spindle shape compared with the hype rtrophic appearance of SMC in the absence of EC. Electron microscopy r evealed EC gap junctions and cytoplasmic projections from SMC of suffi cient length to transverse the pores in the coculture membrane. Conclu sions: This coculture method has several useful features, including an appropriate luminal/abluminal EC/SMC orientation, a short distance be tween the cell layers, the potential for cell-to-cell contact, and the ability to separate the cell types for assays. It is clear that EC ma rkedly affect SMC proliferation, protein/DNA ratio, and structure in c oculture with dynamic interactions occurring for at least 4 days. Thes e effects must be considered when attempting to model in vivo phenomen a in tissue culture.