Figure 6.

Bevacizumab inhibited angiogenesis via macrophage FcγRI and c-Cbl. Bevacizumab suppressed corneal (a) and choroidal (b) angiogenesis in Fcgr1^−/− mice transplanted with wild-type mouse bone marrow, but not in wild-type mice receiving Fcgr1^−/− bone marrow. n=11–16. (c) Bevacizumab and human IgG1 inhibited mouse Vegfa-induced migration, over 12 h, of bone marrow-derived macrophages isolated from wild-type mice but not from Fcgr1^−/−, c-Cbl^−/− or c-Cbl (C379A) mice, which lack E3 ubiquitin ligase activity. n=3. Results are means±s.e.m. *P<0.05 compared with PBS (a–c). (d) Bevacizumab did not inhibit choroidal angiogenesis in NOTAM mice. n=10. (e) Western blot shows induction of c-Cbl phosphorylation in wild-type mouse BMDMs treated with bevacizumab for 15 min. Protein loading was assessed by α-Tubulin abundance. (f) Western blot shows in vivo induction of c-Cbl phosphorylation in wild-type mouse corneas following suture injury that were treated with bevacizumab or its Fc fragment, but not by its Fab fragment. (g) Bevacizumab did not inhibit corneal or choroidal angiogenesis in c-Cbl^−/− mice. n=11–30. NS, no significant difference between groups. (h) Western blots show time-dependent Vegfr1 degradation in wild-type but not NOTAM mouse BMDMs treated with bevacizumab. Protein loading was assessed by HSP70 abundance. (i) Western blots show that RAW264.7 mouse macrophages pre-treated with bevacizumab, but not PBS, 2 h before stimulation with mouse Vegfa, exhibited reduced phosphorylation of PI3K and PLCγ1 at 10 min after Vegfa exposure. Protein loading was assessed by β-actin abundance. (j) Western blots show induction of c-Cbl phosphorylation in human peripheral blood mononuclear cells (PBMC) or in THP-1 human monocytic cells, treated with bevacizumab, but not PBS, for 15 min. Treatment with bevacizumab, but not PBS, reduced VEGFR1 abundance in human PBMCs and THP-1 cells. Protein loading was assessed by Vinculin or α-Tubulin abundance. Images representative of three experiments (e, f, h–j).