Fig. 4. Activation of MMP-1 correlates with increased collagenase activity in ECs during plasma kallikrein- and plasmin-induced capillary tube regression. ECs were suspended in collagen matrices and cultured in the absence (Control) or presence of active plasmin (PL) or kallikrein (Kal) at two doses (0.5 µg/ml, 2.5 µg/ml). Gels were monitored for tube regression and gel contraction via the 384 micro-well regression assay (as in Fig. 3). At the indicated time points, conditioned media were collected and MMP-1 levels were analyzed by western blotting. In addition, conditioned media were analyzed for collagenase activity via the DQ collagenase assay. Triplicate wells of conditioned media from the above time points were incubated with 25 µg/ml of DQ collagen overnight at room temperature. Absorbance was measured at 528±20 nm via a fluorescent microplate reader. Background fluorescence was subtracted prior to reporting of fluorescence in arbitrary units as mean±s.d. (n=3). Time to 50% gel contraction is reported above each data set. In each case, fluorescence corresponded with the degree of MMP-1 activation on the western blot and the time to 50% collagen gel contraction. Arrows and arrowheads indicate the position of MMP-1 in latent or activated forms, respectively.