title: Development of a new in vitro model of elastic fiber assembly in human pigmented epithelial cells.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  These observations indicate that this in vitro model is especially useful for the analysis of mechanisms of elastic fiber formation.
authors.affiliation: Department of Clinical Chemistry, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. wachi@hoshi.ac.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Wachi
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Sato
authors.forename: Hayato
authors.initials: H
authors.lastname: Murata
authors.forename: Junji
authors.initials: J
authors.lastname: Nakazawa
authors.forename: Barry C
authors.initials: BC
authors.lastname: Starcher
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Seyama
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D003895:Desmosine / Q000378:metabolism; D004547:Elastic Tissue / Q000502:physiology*; D005109:Extracellular Matrix / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D020894:Microfibrils / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification; D014333:Tropoelastin / Q000502:physiology*
chemical_list: D011994:Recombinant Proteins; D014333:Tropoelastin; D003895:Desmosine