Fig. 2. Elastin induces a mature contractile phenotype in vascular smooth muscle cells. (A-E) Immunofluorescence analysis for SM -actin reveals that Eln^+/+ vascular smooth muscle cells (A) have a highly organized network of actin stress fibers, a hallmark of mature contractile vascular smooth muscle cells. By contrast, there is a paucity of actin stress fibers in Eln^-/- vascular smooth muscle cells (B; outlined by white dots). The elastin gene product, recombinant tropoelastin, induces the formation of organized actin stress fibers in Eln^-/- vascular smooth muscle cells (D), but does not affect Eln^+/+ vascular smooth muscle cells (C). Scoring analysis demonstrates a significant increase in the percentage of Eln^-/- vascular smooth muscle cells with organized actin myofilaments after elastin treatment (P<0.0001) (E). Tropoelastin mediated actin polymerization is unaffected when Eln^-/- cells are treated with either a gene transcription inhibitor, actinomycin D, or a protein translation inhibitor, cycloheximide. The Rho kinase inhibitor, Y27632, blocks actin polymerization of tropoelastin treated Eln^-/- cells. (F-J) Immunofluorescence analysis for vinculin reveals that Eln^+/+ vascular smooth muscle cells (F) have well-defined focal adhesions (arrowheads). By contrast, Eln^-/- vascular smooth muscle cells (G; outlined by white dots) have poorly defined focal adhesions. Tropoelastin induces well-defined focal adhesions (arrows) throughout Eln^-/- vascular smooth muscle cells (I), but does not affect Eln^+/+ vascular smooth muscle cells (H). Scoring analysis revealed a significant increase (P<0.0001) in the percentage of Eln^-/- vascular smooth muscle cells with defined focal adhesions after tropoelastin treatment (J). (K,L) Immunofluorescence staining with antisera against tubulin reveals no difference between Eln^+/+ vascular smooth muscle cells (K) and Eln^-/- vascular smooth muscle cells (L). Exposure times for all images are the same. Results represent the mean±s.d. from three individual experiments.

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