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Fig. 8. MMP-1 and MMP-10 regulate EC tube regression in three dimensional collagen matrices. (A-C) ECs were transfected with siRNA against MMP-1 or luciferase control. Recombinant adenoviruses were used 24 hours later to deliver GFP or MMP-10. Cultures were established in the absence (A) or presence of 0.5 µg/ml kallikrein (B) or 2 µg/ml plasminogen (C). Gels were monitored every 4 hours for tube regression and percent gel contraction was recorded. At 56 hours, cultures were fixed and conditioned media were collected for western blot analysis of MMP-1 levels (A-C). Note that in all panels a decrease in latent MMP-1 was present in cells treated with MMP-1 siRNA compared to luciferase control. MMP-1 activation occurred due to an increase in MMP-10 levels and also in the presence of the serine proteases plasma kallikrein and plasminogen. (D) ECs were transfected with siRNA against MMP-10 or luciferase control as described. 24 hours later, ECs were infected with GFP-Ad or MMP-1 Ad. Cultures were established in the absence or presence of 2 µg/ml plasminogen. Cultures were monitored every 4 hours for tube regression and percentage gel contraction was recorded. At 56 hours, cultures were fixed and conditioned media were collected for western blot analysis of MMP-10 levels. Note that in all cases, a decrease in latent MMP-10 was present in cells treated with MMP-10 siRNA compared to controls. Arrows and arrowheads indicate the position of MMP-10 in latent or activated forms, respectively.
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