Cytodifferentiation and expression of α-smooth muscle actin mRNA and protein during primary culture of aortic smooth muscle cells. Correlation with cell density and proliferative state

Abstract

To investigate a possible correlation between cytodifferentiation, proliferation, and actin expression, smooth muscle cells from the 9-week-old rabbit aortic media were enzyme-dispersed into single cells and were plated in primary culture at different initial seeding densities. The volume fraction of myofilaments (V(v)myo) in cells seeded moderately densely fell from 39.5% ± 1.2% in the intact aortic media to 11.5% ± 1.6% on Day 5, one day before the onset of logarithmic growth. The V(v) myo remained low over the next 3 days, then began to rise as the density of cells increased, returning almost to the original levels after confluency and 1.84 cumulative population doublings (CPD). The expression of α-smooth muscle actin mRNA followed a similar time course of change, falling from 84.7% ± 1.2% of total actin mRNA in freshly isolated cells to 54.0% ± 6.5% on Day 5, returning to 87.5% ± 0.5% after confluency. In these cultures, the α-smooth muscle actin protein content was 93.7% ± 2.9% of total actin in freshly isolated cells, 68.7% ± 7.3% on Day 5, and 73.3% ± 2.5% 3 days after conlfuency. In densely seeded cultures, the V(v) myo and expression of α-smooth muscle actin mRNA fell only slightly on Day 5 and rose to original levels upon confluency after 0.33 CPD. However, at the protein level, α-smooth muscle actin decreased on Day 5 and remained low on Day 12. The V(v) myo, α-smooth muscle actin mRNA, and actin protein of sparsely seeded cells fell on Day 5 and then remained low throughout the culture period, including 5 days after confluency (Day 24), when the cells had undergone 5.37 CPD. Cells that were maintained subconfluent but quiescent on Day 7 in culture had the same low V(v) myo, low α-sctin mRNA expression, and low α-actin protein content as actively proliferating cells. The results show that V(v) myo and α-smooth muscle actin mRNA undergo parallel changes during primary culture according to seeding density, but not to replication, and that α-smooth muscle actin protein decreases in culture then remains low irrespective of culture conditions.