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

Stephen B Waters, Joseph R Dominguez, Hyun-Dong Cho, Nicolene A Sarich, Asrar B Malik, View ORCID ProfileKaori H Yamada  Correspondence email
Stephen B Waters
1Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, USA
Roles: Data curation, Formal analysis, Investigation
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Joseph R Dominguez
1Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, USA
Roles: Data curation, Formal analysis, Investigation
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Hyun-Dong Cho
1Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, USA
Roles: Data curation, Formal analysis, Investigation
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Nicolene A Sarich
1Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, USA
Roles: Data curation
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Asrar B Malik
1Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, USA
Roles: Supervision, Funding acquisition, Writing—review and editing
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Kaori H Yamada
1Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, USA
2Department of Ophthalmology and Visual Sciences, University of Illinois College of Medicine, Chicago, IL, USA
Roles: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration, Writing—original draft
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  • ORCID record for Kaori H Yamada
  • For correspondence: horiguch@uic.edu
Published 20 October 2021. DOI: 10.26508/lsa.202101170
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    Figure 1. Inhibition of VEGFR2 trafficking prevents vascular leakage and metastasis in mice.

    (A, B) Lung metastasis was examined by pigmented foci of B16F10 melanoma. C57BL/6 received tail vein injection with 1 × 105 of metastatic B16F10 melanoma cells. Mice were 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 14. Treatment of KAI significantly decreased lung metastasis compared with the control peptide. N = 11, 10 for control peptide and KAI, respectively. (C, D) Representative images of H&E and immunostaining of Ki67 of lungs from mice treated with either control peptide or KAI for 2 wk after injection with 1 × 105 B16F10 melanoma via tail vein. Scale bar, 100 μm. Number of Ki67-positive foci/mm2 was measured and shown in graph C as mean ± SEM. (E, F) Evans blue extravasation showing the effect of KAI on VEGF-A-induced endothelial cell permeability in C57BL/6 mice. C57BL/6 received i.v. injection of Evans blue followed by s.c. injection of VEGF-A and KAI or control peptide to test VEGF-A-induced permeability. Extravasation of Evans blue to skin was measured and plotted in a graph. N = 11 and 10 for control peptide and KAI, respectively.

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    Figure 2. Endothelial cell-specific knockout of KIF13B in mice prevents VEGF-A–induced neovascularization and sprouting angiogenesis.

    (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 Kif13btm1c/tm1c endo-SCL-Cre (+) mice, whereas ECs from Kif13btm1c/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 Kif13biECKO and Kif13bWT 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 Kif13biECKO and Kif13bWT 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 Kif13bWT -growth factor (GF), Kif13biECKO -GF, Kif13bWT + VEGF-A, and Kif13biECKO + VEGF-A, respectively. (F, G) VEGF-A–induced EC sprouting from aortic rings of Kif13biECKO and Kif13bWT. The aortic rings were isolated from tamoxifen-treated Kif13biECKO and Kif13bWT 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 Kif13bWT and Kif13biECKO, respectively.

  • Figure 3.
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    Figure 3. Endothelial cell-specific deletion of KIF13B inhibits tumor angiogenesis and tumor growth.

    (A, B, C) Kif13biECKO and Kif13BWT 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).

  • Figure 4.
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    Figure 4. Endothelial specific deletion of KIF13B prevents vascular leakage and metastasis in mice.

    (A, B) Evans blue extravasation showing VEGF-A–induced endothelial cell (EC) permeability in EC-specific Kif13b KO mice. Kif13biECKO and Kif13bWT 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 Kif13bWT and Kif13biECKO, respectively. (C, D) Lung metastasis was examined by pigmented foci of B16F10 melanoma. Kif13bWT and Kif13biECKO 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. Kif13biECKO had a markedly decreased rate of lung metastasis compared to the WT control. N = 18, 16 for Kif13bWT and Kif13biECKO, respectively. (E) Representative image of H&E-stained lungs from Kif13bWT and Kif13biECKO injected with 1 × 105 B16F10 melanoma via tail vein. Scale bar, 100 μm.

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    Figure 5. The peptide KAI targets KIF13B function in endothelial cells.

    (A, B) Cell-type specificity of KAI was examined by lung metastasis of B16F10 melanoma in Kif13bWT and Kif13biECKO. Kif13bWT and Kif13biECKO 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 Kif13bWT+control peptide, Kif13bWT + KAI, Kif13biECKO+control peptide, and Kif13biECKO + KAI, respectively.

  • Figure S1.
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    Figure S1. KIF13B expression in endothelial cells and generation of endothelial cell-specific inducible KIF13B KO mice.

    (A) Scheme of Kif13b WT and a mutant allele. (B-i) Representative image of genotyping shows WT allele using KIF13B-F and KIF13B-R primers (448 bp), FRT cassettes using KIF13B-F and CAS-R primers (251 bp), and LacZ using LacZ-F and LacZ-R primers (108 bp). (ii) Removal of LacZ-neo by FLP generates a tm1c allele showing 661 bp amplicons with KIF13B-F and KIF13B-R primers. Kif13bWT/tm1c heterozygous showed both 661 bp and 448 bp bands. (iii) Kif13btm1c/tm1c homozygous was confirmed by PCR with KIF13B-F and KIF13B-R primers showing only 448 bp band. Endo-SCL-Cre-ERT (+) was confirmed by PCR.

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Prevent metastasis by limiting VEGFR2 trafficking
Stephen B Waters, Joseph R Dominguez, Hyun-Dong Cho, Nicolene A Sarich, Asrar B Malik, Kaori H Yamada
Life Science Alliance Oct 2021, 5 (1) e202101170; DOI: 10.26508/lsa.202101170

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Prevent metastasis by limiting VEGFR2 trafficking
Stephen B Waters, Joseph R Dominguez, Hyun-Dong Cho, Nicolene A Sarich, Asrar B Malik, Kaori H Yamada
Life Science Alliance Oct 2021, 5 (1) e202101170; DOI: 10.26508/lsa.202101170
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Volume 5, No. 1
January 2022
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