Fig. 3

Pharmacological activation of the hypoxia signaling pathway prevents HFD-induced medullary fat accumulation and vascular damage in the skeleton. a BM adipose tissue (BMAT) visualization by toluidine blue staining in tibia sections; lower panels: magnified views of the metaphyseal region below the growth plate. Scale bar, 100 µm. b Adipocyte numbers, as quantified in the metaphysis (n = 7–9 mice/group). c Frequency distribution of adipocyte size measured by area per cell in 2D. d Left panel: single optical image showing a full femur section stained for EMCN (blood vessels, red) and Hoechst (nuclei, blue) depicting the metaphyseal ROI, comprising 1.5 × 1.5 mm, placed under the growth plate; scale bar, 500 µm. Right panels, upper row: maximum intensity projection (MIP) of confocal images of EMCN-stained blood vessels (100 µm depth); scale bar, 200 µm. Lower row: binarized 3D vascular network (white) obtained from semi-automated segmentation of EMCN staining. e–h Morphological analyses of the skeletal vasculature, showing vascularized volume relative to the ROI tissue volume (µm³/µm³ expressed in %) (e); blood vessel surface over tissue volume (µm²/µm³) (f); blood vessel branches (number/µm³ tissue volume), calculated as single strings from node to node in a skeletonized network (g); triple junctions of the vascular network depicting web complexity, calculated as nodes with 3 branches (h). i qRT-PCR of Vegf mRNA expression levels in full humerus extracts, normalized to Hprt. j Analysis of blood vessel diameters, showing (left) representative images of gradient pseudo-coloration from blue to yellow (0–40 µm thick) (scale bar 200 µm) and (right) the frequency distribution of local thickness. k Frequency distribution for the blood vessel branch lengths, considered as lengths of each single string from node to node. l qRT-PCR of Epo levels relative to Hprt expression in humeri harvested from healthy mice. m Epo mRNA levels relative to Hprt expression in bones from vehicle-injected versus FG-4592-treated HFD-fed mice. Statistical analyses were done by two-way ANOVA, with multiple comparisons between diets and treatments; n = 8 (adiposity analyses); n = 3–4 (vascular analyses); n = 7–9 (qRT-PCR analyses). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.000 1; ns not significant