Down-regulation of proangiogenic proteins by EEGS in HUVEC cells. (A) HUVEC cells were incubated in the presence (100 μg/ml) or absence of EEGS for 8 h. The protein expression profiles in the cultured medium and cell lysates were analyzed using a human angiogenesis array kit. The closed and dotted rectangles represent the duplicated EDN1 and FGF2 spots in the proteome membrane chips, respectively. (B) HUVEC cells were treated with increasing concentrations (0–200 μg/mL) of EEGS for 24 h. The amount of EDN1 released into the cultured medium after EEGS treatment was quantified by immunoassay (left). Changes in EDN1 mRNA expression levels were determined by RT-PCR (right). Total RNAs and cDNAs were prepared from HUVEC cells that were treated with increasing concentrations of concentrations of EEGS for 8 h. (C) Changes in MMP2 activity in the HUVEC cultured medium were measured by gelatinolytic zymogram analysis (left). MMP2 activity was observed at the expected molecular weight (67 kDa) on the gelatin-incorporated SDS-PAGE gel. Coomassie staining of a SDS-PAGE gel without gelatin confirmed equal protein loading. The MMP2 active bands were confirmed by western blot analysis. The effect of EEGS on the levels of intracellular MMP2 mRNA was determined by RT-PCR (right). Total RNAs and cDNAs were prepared from HUVEC cells that had been treated with increasing concentrations of EEGS for 8 h. Data are presented as the mean±S.D. of at least three independent experiments. **p<0.01 and ***p<0.001 compared with control treatment.