Effects of collagen gel configuration on behavior of vascular smooth muscle cells in vitro: Association with vascular morphogenesis

The growth, behavior, and contractile protein expression of rabbit aortic s mooth muscle cells (SMC) grown on, between layers, or within a collagen gel was investigated by confocal laser scanning fluorescence microscopy and We stern analysis. SMC grown on collagen gel behaved similarly to those on con ventional culture dishes. However, when a second layer of collagen was over laid, cells underwent an elongated quiescent phase before onset of prolifer ation and a more than threefold lower logarithmic growth rate was observed. These cells self-organized into a network with ring-like structures. With increasing culture time, some of the rings developed into funnel-like, inco mplete or complete tubular structures. If a tubular template preexisted wit hin the gel, the SMC established a cylinder-shaped tube with several circul arly arranged muscular layers (similar to an artery wall). This behavior mi micked endothelial cells during angiogenesis in vitro. A similar phenomenon occurred in cultures in which SMC were randomly mixed in a collagen gel, b ut here their behavior and morphology varied with their position within the gel. Western blot analysis showed that the SMC differentiation marker, smo oth muscle myosin heavy chain-2 (SM-2), rapidly decreased, disappearing by day 10 in SMC grown on collagen, but was still detectable until day 25 in c ells cultured between or within the same gel. These findings indicate that like endothelial cells, vascular SMC can display blood vessel formation beh avior in vitro when an appropriate three-dimensional matrix environment is provided to keep them in a relatively higher-differentiated and low-prolife rative state.