Development of a new in vitro model of elastic fiber assembly in human pigmented epithelial cells

Objectives: We developed an in vitro model of elastic fiber assembly that provides a comparison of the efficiency of different tropoelastin molecules to organize into fibers. Design and methods: Recombinant tropoelastin was added to ARPE-19 cell culture medium. The elastic fiber assembly was evaluated by immunofluorescence staining, the quantitative analysis of cross-linking amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. Results: We confirmed that ARPE-19 cells express fibrillin-containing microfibrils and lysyl oxidase, but they do not express tropoelastin. Immunofluorescence staining showed a dose- and time-dependent increase in the extracellular matrix. The quantity of cross-linking amino acids and matrix-associated tropoelastin also increased together with the matrix-associated elastin. Moreover, the analysis of a radioimmunoprecipitation assay (RIPA) buffer-soluble fraction indicated that tropoelastin interacted with microfibrils and cross-linked elastin was detected as a super molecular complex. Conclusion: These observations indicate that this in vitro model is especially useful for the analysis of mechanisms of elastic fiber formation.