Type IV Collagen Synthesis and Accumulation in Neonatal Rat Aortic Smooth Muscle Cell Cultures

Abstract

The production of type IV collagen by cultured neonatal rat aortic smooth muscle cells was monitored over a three-week period to further characterize the extracellular matrix of this unique culture system. Type IV collagen was quantified using a dot immunobinding assay and was found to represent 1% or less of the total collagen produced by these cells in culture. Total collagen represented up to 33% of the total protein. The pattern of type IV collagen production in the media and the cell layer suggests that although these cells synthesize and secrete type IV collagen from the onset of culture, type IV collagen deposition only occurs after the cells have reached confluence. In the presence of ascorbate the amount of type IV collagen peaked in the media in preconfluent cultuRes In the absence of ascorbate, little type IV collagen was detected in the media. On the other hand, the presence or absence of ascorbate made little difference in the amount of the total collagen detected in the media, although hydroxylation was affected. Remarkably, in the absence of ascorbate type IV collagen accumulation in the cell layer was similar by the end of the culture period to that in cultures treated with ascorbate. Laminin was not affected by the presence or absence of ascorbate. When these cells were exposed to ascorbate for 24 hours, a peak of soluble elastin was detected in the media. However, soluble elastin was not detected in the media in the absence of ascorbate or in cultures which were maintained in the presence of ascorbate. Modulation of the extracellular matrix with ascorbic acid indicated that type IV collagen deposition did not depend on the presence of ascorbic acid and that there was no discernable interaction between type IV collagen, laminin, and elastin.