VEGF down-regulates MMP-9 and inhibits B-CLL cell migration. (A) A total of 3 × 106 B-CLL cells were incubated in RPMI/0.1% fetal bovine serum with the indicated concentrations of VEGF. After 24 hours, the conditioned media was concentrated and analyzed by gelatin zymography. MMP-9 levels on untreated cells were normalized to 1, and average values (n = 7) are shown. (B) B-CLL cells (n = 8) were untreated or treated with 200 ng/mL VEGF or PlGF, and in the presence or absence of 5μM VEGFR2 inhibitor I (R2 inhib.). After 24 hours, MMP-9 was analyzed and quantitated as explained. (C-D) A total of 5 × 105 B-CLL cells (n = 3) were untreated or treated with VEGF or PlGF for 30 minutes and added to Transwell filters (5 μm pore size) coated with Matrigel (A) or activated HUVECs (B) in the presence of the cytokines. Some cells were also pretreated with 5μM VEGFR2 inhibitor I as indicated. CXCL12 (150 ng/mL) was added to the medium in the bottom chamber, except for the control. After 24 hours, migrated cells were counted by flow cytometry. Expression of CD19 on transmigrated cells (> 90%) was also analyzed by flow cytometry. Values are the percentage of total cells added. Bars represent SD. *P ≤ .05, **P ≤ .01, calculated by the 2-tailed Student t test.
