Immunofluorescence microscopy demonstrates a clear increase in collagen and fibronectin production in cells infected with Ad-wt3 compared with cells infected with other viruses. C Western immunoblotting of lysates from infected cells with an antifibronectin antibody middle and an anticollagen I antibody top confirmed increased matrix synthesis by Ad-wt3-infected cells.
The blot was stripped and reprobed with an antibody against HSP70 bottom to confirm equal loading. One representative experiment of three is shown. Percentage of expression of the uninfected cells is set at Immunostaining and Western immunoblotting demonstrated increased synthesis of type I collagen and fibronectin in Ad-wt3-infected cells.
Cells were infected at day 2 after isolation and evaluated at day 7; cells overexpressing wild-type Smad3 but not Smad2 or either dominant negative Smad demonstrated by immunostaining a modest increase in collagen expression and a more marked increase in fibronectin expression compared with uninfected cells Figure 3, A and B. These findings were confirmed by Western immunoblotting Figure 3, C—E. Figure 4. Smad3 enhances chemotaxis of transdifferentiated HSC.
HSC were infected with adenoviruses at day 7 after isolation, as indicated. Cells that had migrated through the inserts 15 h later were stained, photographed, and counted manually. Type I collagen and serum served as chemoattractants, and cells migrating through the inserts were counted. Overexpression of wild-type Smad3 resulted in a significant increase in migration toward serum and type I collagen, whereas dominant negative Smad3 and wild-type and dominant negative Smad2 had no effect Figure 4.
By day 5, these cells seemed to be myofibroblasts, with greater spreading than cells infected with the other viruses. Close examination of Ad-wt3-infected cells at day 8, however, demonstrated that they had a more angular shape, with increased spreading and fewer peripheral protrusions Figure 5B ; also shown in Figure 5D , top.
Note in the top panels that the shape of infected cells is significantly different and that the entire population of infected cells is affected; in bottom panels, both an increase in size and number of focal adhesions is evident. Total levels of FAK were unchanged, as assayed by Western blotting our unpublished data. When activated HSC were infected at day 7, they remained activated for at least 10 d, even when infected with the dominant negative Smads.
All other HSC uninfected or infected with any of the other adenoviruses had phenotypic appearances identical to each other, with prominent membrane protrusions and without the angular shape that characterized the Ad-wt3-infected-cells.
Cells coinfected with two viruses demonstrated either no change from uninfected or, if infected with Ad-wt3, demonstrated increased focal adhesions and stress fiber organization Figure S1. To determine whether Smad3 expression alone is sufficient to induce activation of HSC, primary HSC were plated on the basement membrane-like matrix Matrigel immediately after isolation and were then infected with adenoviruses at day 2.
Culture on Matrigel is a well established model for maintaining HSC in the quiescent state and for causing reversion of activated cells to quiescence Friedman et al. All cells remained quiescent except cells infected with Ad-wt3, which surprisingly seemed to migrate through the thin layer of Matrigel, and, after reaching the glass or plastic support below, underwent activation Figure 7 and Video 3.
A similar phenomenon also was observed when activated HSC were infected with adenoviruses on day 7 and passaged onto Matrigel on day 8 our unpublished data. The mechanism whereby Ad-wt3-infected HSC migrates through Matrigel is not known although it likely involves a protease. Figure 5. A Infected HSC were lysed at days 3, 5, and 7 after isolation, as labeled. B Light microscopic analysis of cells at day 6 after isolation demonstrates significant morphological differences between cells infected with Ad-wt3 and cells infected with other viruses or uninfected cells; our unpublished data.
The Smad3-overexpressing cells display a more angular shape, fewer membrane protrusions, and an obvious increase in cytoskeletal organization.
We overexpressed wild-type and dominant negative Smads2 and 3 in primary HSC using adenoviral vectors. Our data demonstrate that Smad3 is a direct mediator of HSC matrix production and matrix interactions. Smad3 increased expression of collagen and fibronectin, without a significant effect on proliferation. Smad2, on the other hand, seemed to be primarily antiproliferative, with a minimal role in HSC fibrogenesis.
Evidence from other model systems also implicates Smad3 as the dominant Smad in extracellular matrix deposition, wound healing, and fibrosis Verrecchia et al.
Smad3 null mice are less susceptible to bleomycin-induced lung fibrosis and radiation-induced skin fibrosis than wild-type mice Flanders et al. Similarly, in the liver, collagen expression in response to an acute fibrogenic stimulus was decreased by about one-half in Smad3 null mice compared with their wild-type counterparts Schnabl et al. A number of genes important in fibrogenesis, including several collagen genes and regulators of matrix degradation, seem to be Smad3- but not Smad2-dependent Verrecchia et al.
These findings are in contrast to one report that Smad2, not Smad3, is important in the transdifferentiation of pancreatic stellate cells, which are similar to HSC. In their study, however, Ohnishi et al. Lovell L. Sandrine O'Brien Caragh M. Shen L. Tudor C. M Zinck Valija Zito V. Vive Le Douanier Rousseau! Entre Terre Et Ciel. Special Forces officer Rebecca Hart Add the first question.
Start your free trial. Le Disque des Vacances. Esmeralda is an inexperienced director, but she dreams of this film where everything will be heard, where everyone can express their point of view without risk of being interrupted.
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