Fig. 8. Recombinant human ANG (rhANG) rescues GC-impaired bone growth and mineral acquisition.

Three-week-old BALB/c mice were treated with vehicle, MPS alone at 10 mg/m²/day or MPS plus rhANG (1 µg/day) by daily intraperitoneal injection for 4 weeks. Representative images of SA-βGal staining (white) and immunofluorescence staining of Endomucin (Emcn, red) in primary spongiosa of femoral bone in (a). Percentage of SA-βGal-expressing vessels were quantified in (b). Double immunofluorescence staining of Emcn (red) and CD31 (green) in metaphysis of femoral bone in (c). DAPI-stained nuclei blue. Relative yellow fluorescence intensity in primary spongiosa was measured in (d). Immunofluorescence staining of femoral metaphysis sections were performed using antibody against Osx (red) in (e). DAPI stains nuclei blue. Numbers of Osx⁺ cells per mm² tissue area (N. Osx⁺ cells/Ar) were quantified in (f). Representative μCT images of distal femur in mice were shown in (g, longitudinal sections) and (h, cross sections). Quantitative analyses of trabecular bone volume fraction (BV/TV) (i), trabecular thickness (Tb. Th) (j), trabecular number (Tb. N) (k), and trabecular separation (Tb.Sp) (l). Trichrome staining of the metaphyseal trabecular bone at distal femora in (m). Osteoid stains red and mineralized bone stains green. Osteoid surface per bone surface (OS/BS) was measured in (n). Representative images of calcein double labeling (o) and quantification of bone formation rate per bone surface (BFR/BS) (p) of the metaphyseal trabecular bone at distal femora. GP growth plate. n = 6-10 mice. Data are represented as mean ± s.e.m. **p < 0.01 as determined by one-way ANOVA with post hoc Tukey test.