Hypoxia-induced signaling for neovasculogenesis is highly enhanced in SCD mouse BM. (A) Representative 2D images of femoral diaphyses from AA and SS mice created using LaSC. Whole-femoral sections of 12-week-old AA or SS mice were stained with endoglin and HIF-1α, followed by LaSC analysis. Scale bars, 100 μm. (B) DAPI⁺ HIF-1α^hi cells were quantitated by LaSC. The bar graph shows the percentage of HIF-1α^hi cells in the BM as mean + standard error of the mean (SEM), n = 4 mice per group (12-15 weeks old; 2 female mice and 2 male mice). (C) Hypoxia-induced signaling was analyzed by western blot, followed by densitometric analysis in the digested BM (dBM) cells from hindlimbs. The proteins in the BM exudates or cell lysates were resolved by 4% to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels, followed by immunoblotting with specific antibodies, as indicated, and β-actin antibody for loading controls. Representative blots are shown. Fold change (Δ) in band intensity of proteins and p44/42 MAPK phosphorylation were normalized to β-actin and p44/42 MAPK protein loading, respectively, and shown as mean ± SEM (n = 4 each; 12-15 weeks old; 2 female mice and 2 male mice). (D) Immunoblot (IB) analysis with specific antibodies against VEGF-A, Ang 1, Ang 2, and VCAM-1 of BM exudates from AA and SS mice (left panel). Immunoglobulin G (IgG) light chain (L chain) was used as a loading control. One representative gel of 6 with similar results is presented. Densitometric analysis of immunoblots is shown as a bar graph (right panel). Results are shown as protein intensity relative to IgG. Ang 2 represents immature and mature Ang 2 forms. Data are presented as mean + SEM (n = 6 from each strain; 12-15 weeks old; 3 female mice and 3 male mice). *P < .05, **P < .01, ***P < .001, Student t test. AU, arbitrary units; M.W., molecular weight.