Figure 7
Choroidal, Matrigel-supported and tumor neovascularization in Sema6A−/− and wild-type mice. (A) Choroidal neovascularization was evaluated on day 7 after laser injury to the Bruch membrane; representative images of injured areas in representative wild-type and Sema6A-null littermates (6 weeks old); isolectinB4 staining. (B) Quantitative analysis of choroidal neovascularization 7 days after laser injury to the Bruch membrane in groups of wild-type and Sema6A-mutant mice (5 to 6 weeks old). The area occupied by vessels was measured after staining eye wholemount with isolectin B4; the results reflect the group means (n = 8; mean ± SEM; p reflects the statistical significance of group difference). (C) Matrigel plugs containing VEFGA (150 ng/mL), FGF2 (150 ng/mL), and heparin (0.5 mg/mL) were produced subcutaneously in wild-type and in Sema6A−/− mice (5 weeks old); plugs were removed after 7 days, processed for histology, and stained for CD31 and NG2. Representative images: the arrow points to a vessel within the matrigel plug, magnified in the inset. (D) Quantative analysis of matrigel neovascularization in groups of wild-type and in Sema6A−/− mice (5 to 6 weeks old; 5/group) based on CD31 positive area. (E) Sema6A immunostaining of representative Matrigel plugs from wild-type and Sema6A−/− mice. Nuclei are detected with DAPI. Corresponding bright field images are shown in the top panels. (F) B16 murine melanoma and (G) LLC/1 Lewis lung murine carcinoma cells (10 × 106) were injected subcutaneously in groups of wild-type and Sema6A−/− mice. The tumors were removed after 10 days (B16) or 12 days (LLC/1) and weighed. The bar graphs represent the mean (n = 14/group; +/− SEM) tumor weight/group. CD31 and NG2 immunostaining in B16 (H-I) and LLC/1 (J-K) tumor tissues. Representative images of immunostaining are shown in (H) and (J). The bar graphs in (I) and (K) represent the mean CD31 staining (area of CD31 staining/unit area of tumor; n = 14/group; +/− SEM). The P values reflect the statistical significance of group differences.

Choroidal, Matrigel-supported and tumor neovascularization in Sema6A−/− and wild-type mice. (A) Choroidal neovascularization was evaluated on day 7 after laser injury to the Bruch membrane; representative images of injured areas in representative wild-type and Sema6A-null littermates (6 weeks old); isolectinB4 staining. (B) Quantitative analysis of choroidal neovascularization 7 days after laser injury to the Bruch membrane in groups of wild-type and Sema6A-mutant mice (5 to 6 weeks old). The area occupied by vessels was measured after staining eye wholemount with isolectin B4; the results reflect the group means (n = 8; mean ± SEM; p reflects the statistical significance of group difference). (C) Matrigel plugs containing VEFGA (150 ng/mL), FGF2 (150 ng/mL), and heparin (0.5 mg/mL) were produced subcutaneously in wild-type and in Sema6A−/− mice (5 weeks old); plugs were removed after 7 days, processed for histology, and stained for CD31 and NG2. Representative images: the arrow points to a vessel within the matrigel plug, magnified in the inset. (D) Quantative analysis of matrigel neovascularization in groups of wild-type and in Sema6A−/− mice (5 to 6 weeks old; 5/group) based on CD31 positive area. (E) Sema6A immunostaining of representative Matrigel plugs from wild-type and Sema6A−/− mice. Nuclei are detected with DAPI. Corresponding bright field images are shown in the top panels. (F) B16 murine melanoma and (G) LLC/1 Lewis lung murine carcinoma cells (10 × 106) were injected subcutaneously in groups of wild-type and Sema6A−/− mice. The tumors were removed after 10 days (B16) or 12 days (LLC/1) and weighed. The bar graphs represent the mean (n = 14/group; +/− SEM) tumor weight/group. CD31 and NG2 immunostaining in B16 (H-I) and LLC/1 (J-K) tumor tissues. Representative images of immunostaining are shown in (H) and (J). The bar graphs in (I) and (K) represent the mean CD31 staining (area of CD31 staining/unit area of tumor; n = 14/group; +/− SEM). The P values reflect the statistical significance of group differences.

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