A Controlled Precursor Add-Back Model of Elastogenesis in Smooth Muscle Cell Cultures

Abstract

Neonatal rat aortic smooth muscle cell cultures are capable of synthesizing and accumulating relatively large amounts of insoluble elastin in the extracellular matrix. There are two major soluble elastin molecules in these cultures, one of 77kDa (protropoelastin) and the other of 71 kDa (tropoelastin). We examined the ability of the cell culture system to insolubilize exogenously added soluble elastin precursor moieties into the elastin matrix. To accomplish this, cultures were allowed to develop an enriched elastic fiber matrix for approximately two weeks in first passage. This accumulated matrix then served as the “substrate” for the exogenously added precursor elastin molecules. Culture-derived radioactive soluble elastin was added to the “substrate” cultures and the presence of radioactivity in the insoluble elastin as well as in the lysinederived crosslinks unique to elastin (desmosines) was measured. When purified [3H]-valine radiolabeled protropoelastin was used, more than 15% of the radioactivity added was detected in the alkali-resistant insoluble elastin within 24 hours. After an initial 4-hour incubation of the cells with [3H]-lysine-labelled soluble elastin, most of the radioactivity in the insoluble elastin was associated with the lysine and only a negligible amount was detected in the desmosines. However, during a 16-day chase period, the ratio of radioactive desmosines to lysine increased dramatically, suggesting that not only insolubilization, but crosslinking occurs as well. The addback system described herein should provide a means to probe the molecular properties of protropoelastin and increase our understanding of the mechanisms of elastic fiber formation.