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Fig. 2. Immobilized Tat promotes adhesion and motogenic activity in ECs. (A) GM7373 cells were allowed to adhere to plastic coated with the indicated concentrations of native GST-Tat (
), GST (
), FN (
) or BSA (
). (B) GM7373 cells were incubated for 1 hour at 4°C with the indicated concentrations of anti-
vß3 neutralizing monoclonal antibody LM 609 (closed symbols) or irrelevant IgG (open symbols). Then, cells were allowed to adhere to plastic coated with 20 µg/ml of GST-Tat (circles) or FN (squares). (C) GM7373 cells were allowed to adhere to plastic coated with 20 µg/ml of native (a) or heat denatured (e) wild-type GST-Tat, GST-Tat
1-21 (b), GST-TatK49/52/53/55/56/57A (c), GST-Tat-1e (d). (D) HUVE cells or MAE cells were allowed to adhere on plastic coated with 20 µg/ml of GST-Tat (black bars), FN (hatched bars), or BSA (white bars). At the end of incubation, cell adhesion was quantified. In B, data are expressed as the percentage of cells adherent to the different substrata in the absence of antibodies. Each point is the mean ± s.e.m. of three or four determinations in duplicate. (E) GM7373 cell monolayers adherent to the indicate plastic-immobilized proteins or to tissue culture plastic (t.c.p.) were wounded and incubated for 48 hours in culture medium containing 0.4% FCS in the absence (white bars) or in the presence of free GST-Tat (100 ng/ml; black bars), cRGDfV (grey bars) or cRADfV (hatched bars) (both at 3 µM). At the end of incubation the area of the wound was quantified by image analysis. Each point is the mean ± s.e.m. of four to five fields measured in one experiment out of two or three that gave similar results. The results are expressed as percentage of repair in respect to untreated monolayers adherent to immobilized Tat. (F) Representative microphotographs (magnification 50x) of Tat-adherent GM7373 cell monolayers at T0 and 48 hours after the wounding.
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