KIF13B-mediated VEGFR2 trafficking is essential for vascular leakage and metastasis in vivo

(A) Western blots showing expression of endogenous KIF13B in mouse pulmonary endothelial cells (mouse EC) and HUVEC. Hepatocyte HepG2 was used as a control. (B) Real-time PCR revealed that KIF13B expresses 186 ± 56, 175 ± 28, 49 ± 22 copies/ng RNA in HUVEC, HepG2, and human smooth muscle cells, respectively. N = 9, 9, 3, respectively. (C) Western blotting showing deletion of KIF13B in ECs from tamoxifen-treated Kif13b^{tm1c/tm1c endo-SCL-Cre (+)} mice, whereas ECs from Kif13b^tm1c/tm1c control mice express KIF13B. ECs were isolated from mice lungs using anti-CD31 antibody and anti-ICAM2 antibody (Jin et al, 2012). The expression of KIF13B (250 kD band, arrowhead) was detected by anti-KIF13B Ab. CD31⁻ cells also show a truncated KIF13B band. Α-smooth muscle actin is a marker of smooth muscle cells and myofibroblast. Isolated ECs (CD31⁺/ICAM2⁺ population) from both mice were CD31⁺ and smooth muscle actin. (D, E) VEGF-A–induced neovascularization in Matrigel plug in Kif13b^iECKO and Kif13b^WT mice. After tamoxifen treatment, Matrigel supplemented with 40 ng/ml (1.7 nM) VEGF-A and 2 U heparin, or 2 U heparin alone (-growth factor) were injected s.c. in Kif13b^iECKO and Kif13b^WT counterpart mice. 7 d after implantation, the Matrigel plugs were collected and stained with anti-CD31 Ab. (A) Representative images were shown in (A). Scale bar: 100 μm. (B) The number of CD31-positive vessels was counted and shown as mean ± SE in graph (B). N = 3, 4, 6, and 6 for Kif13b^WT -growth factor (GF), Kif13b^iECKO -GF, Kif13b^WT + VEGF-A, and Kif13b^iECKO + VEGF-A, respectively. (F, G) VEGF-A–induced EC sprouting from aortic rings of Kif13b^iECKO and Kif13b^WT. The aortic rings were isolated from tamoxifen-treated Kif13b^iECKO and Kif13b^WT and embedded in the collagen gels supplemented with VEGF-A. (C) VEGF-A–induced sprouting was analyzed as shown in (C). Scale bar: 200 μm. (G) The number of sprouting was shown as mean ± SE in (G). N = 7 and 6 for Kif13b^WT and Kif13b^iECKO, respectively.

(A, B, C) Kif13b^iECKO and Kif13B^WT mice were treated with tamoxifen i.p. an injection every second day for 2 wk. 3 wk after the completion of tamoxifen injections, luciferase-expressing B16F10 were injected s.c. at the right flank of these mice. (A, B) Tumor growth was monitored by bioluminescence using the IVIS imaging system, the representative images of day 12 were shown in (A), and the graph with mean ± SE was shown in (B). N = 6. t test, P = 0.0018. In (C), tumor growth was also monitored by measuring with a caliper, and shown in the graph by mean ± SE. N = 6. (D, E) The tumor was isolated at day 12 and stained with CD31 to visualize vascularization in the tumor. (D) The representative images were shown in (D). Scale bar: 100 μm. (E) The number of CD31 positive capillaries were counted and shown as mean ± SE in graph (E).

(A, B) Evans blue extravasation showing VEGF-A–induced endothelial cell (EC) permeability in EC-specific Kif13b KO mice. Kif13b^iECKO and Kif13b^WT received i.v. injection of Evans blue followed by s.c. injection of VEGF-A or PBS to test VEGF-A-induced permeability. Extravasation of Evans blue to skin was measured and plotted in a graph. N = 13 and 9 for Kif13b^WT and Kif13b^iECKO, respectively. (C, D) Lung metastasis was examined by pigmented foci of B16F10 melanoma. Kif13b^WT and Kif13b^iECKO received tail vein injection with 1 × 10⁵ of metastatic B16F10 melanoma cells. 2 wk after injection, the lungs were isolated and fixed. The pigmented foci on the lung surface were examined. The plots show the number of metastatic foci in the lung on day 13. Kif13b^iECKO had a markedly decreased rate of lung metastasis compared to the WT control. N = 18, 16 for Kif13b^WT and Kif13b^iECKO, respectively. (E) Representative image of H&E-stained lungs from Kif13b^WT and Kif13b^iECKO injected with 1 × 10⁵ B16F10 melanoma via tail vein. Scale bar, 100 μm.

(A, B) Cell-type specificity of KAI was examined by lung metastasis of B16F10 melanoma in Kif13b^WT and Kif13b^iECKO. Kif13b^WT and Kif13b^iECKO received tail vein injection with 1 × 10⁵ of metastatic B16F10 melanoma cells. Each genotype of mice was divided into two groups; each group received injections of control peptide or KAI (10 mg/kg b.w.) three times/week for 2 wk, and the lungs were isolated and fixed. The pigmented foci on the lung surface were examined. The plots show the number of metastatic lung foci on day 19. Treatment of KAI significantly decreased lung metastasis compared with the control peptide. N = 8, 7, 5, and 5 for Kif13b^WT+control peptide, Kif13b^WT + KAI, Kif13b^iECKO+control peptide, and Kif13b^iECKO + KAI, respectively.