Figure 2

Angiogenesis is suppressed in implanted sponges in α-KO mice.

(a–c) Immunohistochemical analysis of blood vessel formation in implanted sponge. (a) H&E-stained cross-sectional area of implanted sponges at day 14 after implantation (upper row) and immunohistochemistry of CD31 (brown) in the adjacent sections (bottom row). Boxed areas in (a) show as high magnification panels in (b) with the vascularized areas delineated by dotted lines. Using Flox mice as the control (a, left), CD31 positive vascularized areas in α-KO (a, right) were examined in male (c, left) and female (c, right) on days 14 and 28 after implantation. n = 4–5 mice per group. (d) Double immunostaining of CD31 (brown) and Ki67 (black) and counterstaining with hematoxylin (pale blue) in ingrowing connective tissue. The density of CD31-positive blood vessels is higher in the Flox (left) as compared with that in the α-KO (right) mice. (e–g) Morphometric analysis of angiogenesis and endothelial cell proliferation in connective tissues. Number of blood vessels in vascularized areas (e), Ki67 positivity in CD31-positive endothelial cells (f) and cumulative percentages of blood vessel diameters (g) of both genotypes; n = 4 per group. (h) Real-time PCR analyses of mRNA expression levels of endothelial cell-related gene Cldn5, Cdh5 and Cd31 in sponges of Flox and α-KO mice. Male mice were analyzed in (d–h). *p < 0.05 versus Flox mice; **p < 0.01 versus Flox mice; ***p < 0.001 versus Flox mice. Scale bars indicate 1 mm (a), 500 μm (b) and 100 μm (d).