Purpose: Smooth muscle cell (SMC) growth kinetics are often studied in culture without consideration of endothelial cell (EC) influences that occur in vivo. This study examined the time-dependent effect of EC on 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 microns thick polyethylene terephthalate membrane. SMC were studied in coculture opposite from confluent EC (EC/SMC). Controls included SMC cultured opposite SMC (SMC/SMC) or SMC alone (with no cells on the opposite side of the membrane, phi/SMC). After cocultures were established, SMC were harvested from 1 to 4 days after release from growth arrest (n = 5 cultures/day/group). SMC DNA and protein content and 3H-thymidine incorporation were measured in each group. SMC proliferation was indexed by 3H-thymidine incorporation per cellular DNA content.
Results: EC stimulated SMC proliferation 56% more than SMC/SMC cultures and 244% more than SMC alone on day 1 after growth arrest (p < 0.05). This effect decreased with time so that by day 4, EC seemed to inhibit SMC proliferation (49% less proliferation than SMC/SMC and 76% less than SMC alone, p < 0.05). SMC opposite EC had significantly less protein/DNA than control SMC, and they retained a thin, spindle shape compared with the hypertrophic appearance of SMC in the absence of EC. Electron microscopy revealed EC gap junctions and cytoplasmic projections from SMC of sufficient length to transverse the pores in the coculture membrane.
Conclusions: This coculture method has several useful features, including an appropriate luminal/abluminal EC/SMC orientation, a short distance between 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 markedly affect SMC proliferation, protein/DNA ratio, and structure in coculture with dynamic interactions occurring for at least 4 days. These effects must be considered when attempting to model in vivo phenomena in tissue culture.