Effect of MMP9-induced HSPB1 cleavage on VEGF-mediated endothelial cell activation.

<p><b>A</b> HUVECs infected with MMP9 shRNA lentiviral and control vectors were pretreated for 30(1ug/ml) or rHSPB1 fragments (59–203; 1 µg/ml) and then incubated with VEGF (30 ng/mL). After 10 min, cell lysates and conditioned media were subjected to western blotting using indicated antibodies. Bar graph shows ± SEM of band intensities of P-VEGFR2 signals normalized to P-VEGFR2 signals of control for 3 independent experiments. <b>B</b> After 48 hr, proliferation of HUVECs was analyzed by MTT assay. <b>C</b> Transendothelial migration of HOS cells in the presence of rHSPB1 WT (1ug/ml) or rHSPB1 fragments (59–203; 1 µg/ml). After HOS cells were marked with CytoTracker, they were allowed to attach and migrate to the HUVEC monolayer. Fluorescence graph of migratory cells labeled with CytoTracker shows ± SEM for three independent experiments (*<i>P</i> <0.05 and **<i>P</i> <0.01, upper graph). HUVECs infected with MMP9 shRNA lentiviral and control vectors were subjected to tumor transendothelial migration assay in the presence of rHSPB1 WT (1ug/ml) (bottom graph). <b>D</b> HUVECs infected with MMP9 shRNA lentiviral and control vectors were transfected with sHSPB1WT or sHSPB1 fragments (1–58, 59–203) and then activated with VEGF (30 ng/mL). After 10 min, cell lysates and conditioned media were subjected to western blotting. Bar graph shows ± SEM of band intensities of P-VEGFR2 signals normalized to P-VEGFR2 signals of control for 3 independent experiments. <b>E</b> After 48 hr, proliferation of HUVECs was analyzed by MTT assay.