Fig. 5: Adipocyte ESRRA deficiency rescues type H vessel formation under osteoporotic condition by facilitating SPP1-induced angiogenesis.
From: Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow
a Representative images of metaphyseal type H vessels near growth plate immunostained for Endomucin (EMCN, red) and CD31 (green) in distal femurs of Esrrafl/fl and EsrraAKO mice following sham and OVX. DAPI (blue) is used for counterstaining of nuclei (scale bar: 100 μm). b Quantification of CD31+ EMCN+ type H vessel intensity per mm2. n = 7 mice per group. c Immunostaining of Osterix (red) with DAPI (blue) in the metaphysis of distal femurs of Esrrafl/fl and EsrraAKO mice following sham and OVX (scale bar: 50 μm). d Quantification of Osterix+ cells in bone marrow per mm2. n = 7 mice per group. e Schematic diagram showing the procedure of the conditioned medium (CM) preparation, tube formation assay and cell migration assay (schematic created with BioRender.com. Agreement number: VY26KB8O73). The concentrations of soluble SPP1 in BMAds-CM (f) or gWAT-CM (g) prepared from Esrrafl/fl and EsrraAKO OVX mice were measured by ELISA. n = 6, 4 biologically independent samples, respectively. Microvascular endothelial cells (ECs) migration in response to BMAds-CM (h) or gWAT-CM (i) with the addition of 0.5 μg/ml recombinant SPP1 (rSPP1), 1 μg/ml neutralizing SPP1 antibody (SPP1 Nab), or an equal volume of IgG for 24 h was followed by quantification and presentation of representative images from four independent experiments (scale bars: 100 μm). n = 4 biologically independent experiments. j–m Matrigel tube formation assay was performed using ECs and BMAds-CM (j) or gWAT-CM (l) with the indicated treatments for 4 h (scale bar: 100 μm). The length of the tubes and the number of branch points (k, m) per field were analyzed. n = 4 biologically independent experiments. Data are shown as mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001. Statistical analysis is performed using unpaired two-tailed Student’s t test (f, g) and two-way ANOVA with Fisher’s LSD post hoc analysis (b, d, h, i, k, m). Source data are provided as a Source Data file.
