Figure 1.

Increasing S1P levels by pharmacological inhibition of the S1P lyase or genetic deletion of the enzyme increases bone vascular density and promotes pro-osteogenic H-type endothelial cell formation. (A) S1P plasma levels of C57BL/6J mice after 3 and 6 weeks (wk) of DOP treatment (3 mg/kg/d) and untreated controls (n = 12/8/12), bone marrow (BM) S1P levels and BM/Plasma S1P ratios of these mice (n = 4/5/4) (B) quantification of vascular area per total bone marrow area in femoral bone (n = 12/12/6) and mean fluorescence intensity (MFI) of endomucin-stained vessels within the metaphyseal area (n = 7/6/3), (C) representative images; scale bars = 500 μm (top) and 200 μm (bottom). (D) Representative images and gating strategy of CD45⁻, CD31⁺, and Emcn⁺ bone marrow endothelial cells as analyzed by flow cytometry, (E) quantification of endothelial CD31 MFI, endomucin MFI, Emcn^HI/CD31^HI H-type endothelial cells per bone, and percentage of total bone marrow cells (n = 5/6), (F) representative images of the vasculature within the femoral bone of Sgpl^flox/flox Cre⁻ and Sgpl^flox/flox Cre⁺ mice (scale bars = 500 μm [top] and 200 μm [bottom]) and (G) quantification of vascular area per bone marrow area (n = 11/7). Data are presented as mean ± SEM, one-way ANOVA (A and B), or 2-tailed t-test (E and G) were used for statistical analysis.