In-Situ Hybridization of Tropoelastin mRNA during the Development of the Multilayered Neonatal Rat Aortic Smooth Muscle Cell Culture

Abstract

Cultured neonatal rat aortic smooth muscle cells are active in synthesizing and depositing large amounts of elastin in their extracellular matrix, making this an ideal system for studying elastogenesis. In this study, the ability of individual cells to synthesize tropoelastin was examined by in-situ hybridization methods. One-micron semi-thin epoxy resin-embedded transverse sections of cells cultured 1, 2, 3 and 4 weeks showed an increase with time in both the number of cells with hybridization signal and the signal intensity; tropoelastin mRNA hybridization signal intensity decreased thereafter up to 8 weeks in culture. In longitudinal sections through the early cultures (1-week), we observed mitotic cells with no detectable hybridization signal, and non-mitotic cells with either no, little or high signal intensity. These data suggest that mitotic cells do not synthesize tropoelastin, and that there is a strong correlation between the hybridization signal intensity and the rate of tropoelastin synthesis. These data also suggest in-situ hybridization methods can detect which cell(s) contain tropoelastin mRNA, their location in the multilayer, and variations in signal intensity. We conclude it is possible to correlate hybridization signal intensity with variations of tropoelastin mRNA levels within individual cells of the cultured smooth muscle cell multilayer.