Building elastin - Incorporation of recombinant human tropoelastin into extracellular matrices using nonelastogenic Rat-1 fibroblasts as a source forlysyl oxidase

The purpose of this study was to assess the feasibility of crosslinking exo genously produced tropoelastin, the precursor of insoluble elastin, into ex isting elastin, Tritiated recombinant human tropoelastin (rhTE) was added t o neonatal rat aorta smooth-muscle cell (NNRSMC) cultures. As much as 12% o f the added rhTE was incorporated into the NNRSMC-derived insoluble elastin with the formation of the elastin crosslinks desmosine (DES) and isodesmos ine (IDES) in a time-dependent fashion. The ratio of radioactivity found in DES and IDES crosslinks to that found in lysyl residues increased from 0.1 8 immediately after incubation with rhTE to 0.76 after 14 d. Crosslinking o f rhTE into elastin and the accompanying formation of tritiated water was i nhibited by beta -aminoproprionitrile, a potent inhibitor of lysyl oxidase, an enzyme critical for the post-translational processing of elastin and co llagen. Acellular NNRSMC matrices were produced and replated with Rat-1 fib roblasts, cells that were found to express lysyl oxidase but not tropoelast in. At 14 d after incubation with rhTE, the ratio of DES and IDES radioacti vity to that of lysine in the insoluble elastin was 0.38. We show for the f irst time that cells expressing lysyl oxidase, but not elastin, as well as elastogenic cells can incorporate rhTE into insoluble elastin with the form ation of elastin crosslinks, This novel approach might be used to augment e lastin repair In certain pathologic states.