CCL1 induces pro-MMP2 in human vascular smooth muscle cells. VSMCs were incubated with DMEM or with CCL1 (150 ng/mL) for 6 hours. The samples were analyzed by gelatinolytic zymography as detailed in “Materials and methods.” (A) CM from VSMCs incubated with DMEM (lanes 1,3) or with CCL1 (lanes 2,4) was tested in duplicate for gelatinolytic activity. CM from CCL1-treated VSMCs showed enhanced gelatinolytic activity. (B) Gelatinolytic zymograms from 3 separate experiments were quantified using the NIH Image 1.61f program. CCL1 increased VSMC gelatinolytic activity by 187%, 250% (samples of panel A), and 152%, respectively, over the untreated controls. (C) CM was concentrated approximately 16-fold using heparin-agarose beads as detailed in “Materials and methods.” Samples containing equal amounts of protein were analyzed by gelatinolytic zymography. MMP-9 standard (1); protein standards phosphorolase-B (107 kDa), albumen (90 kDa) (2); VSMCs stimulated with TNF-α (20 ng/mL) (3), DMEM (4) or CCL1 (5) (150 ng/mL) for 6 hours. TNF-α increased MMP-9 gelatinolytic activity in VSMCs compared with the media control but CCL1 did not. These studies show that CCL1 increases the secretion of MMP-2 by VSMCs but not the secretion of MMP-9.