MMP-12 releases su-PAR fragments from and is expressed in hMVECs in vitro.

(A) Confluent hMVEC cultures, stimulated with the combination of bFGF (50 ng/mL) and VEGF-A (25 ng/mL) to increase u-PAR expression, were washed 3 times with M199 medium supplemented with 0.01% HSA and incubated with the indicated (active) recombinant MMPs (100 ng/mL in M199, 0.01% HSA) for 2 to 4 hours. The supernatants were collected, and the amount of released su-PAR fragments was determined using a su-PAR ELISA and expressed as mean ± SEM (in picograms per 10⁵ cells) of triplicate wells. The data are representative of 3 independent experiments. (B) The mRNAs of the Northern blot experiment of Figure 3 were used for RT-PCR analyses of MMP-12 expression. PCR amplification was obtained after 40 cycles as described by Konttinen et al.52 The + indicates MMP-12⁺ mRNA of synovial fibroblasts; -, negative control (all reagents without template); C, control hMVECs; and bT, bFGF/TNF-α–stimulated hMVECs.