Osteocyte mitochondria regulate angiogenesis of transcortical vessels

Abstract

Transcortical vessels (TCVs) provide effective communication between bone marrow vascular system and external circulation. Although osteocytes are in close contact with them, it is not clear whether osteocytes regulate the homeostasis of TCVs. Here, we show that osteocytes maintain the normal network of TCVs by transferring mitochondria to the endothelial cells of TCV. Partial ablation of osteocytes causes TCV regression. Inhibition of mitochondrial transfer by conditional knockout of Rhot1 in osteocytes also leads to regression of the TCV network. By contrast, acquisition of osteocyte mitochondria by endothelial cells efficiently restores endothelial dysfunction. Administration of osteocyte mitochondria resultes in acceleration of the angiogenesis and healing of the cortical bone defect. Our results provide new insights into osteocyte-TCV interactions and inspire the potential application of mitochondrial therapy for bone-related diseases.

Fig. 1. Osteocytes connect to endothelial cells via dendrites.

a Representative SEM images from 2-month-old male WT mouse femur cortical bone revealed abundant dendritic (yellow arrow) connections from osteocytes with inflated endfeet abutting TCVs. Scale bars, 5 µm. b Representative TEM images from 2-month-old male WT mice showing the dendrite connections (yellow arrow) between lacunae-chambered osteocytes and endothelial cells in the vessel canals. Scale bars, 10 µm. c Representative confocal images of femur cortical bone from three 4-week-old male WT mice injected with Evans Blue intraperitoneally showed multiple dendrites extending from osteocytes to blood vessels. d Representative confocal images of femur cortical bone from 4-week-old male WT mice showed the dendrite connection between osteocytes and endothelial cells. Scale bars, 20 µm. e Schematic diagram of osteocytes beside TCVs for further analysis and quantification. This figure was created by P.L. and cartonized by Ms. Lina Cao. f Pie diagram of the dendrites of osteocytes beside TCV of d (n = 119 osteocytes examined over 5 independent mice). g Percentage of the dendrites on the vessel-offside or vessel-nearside among all the dendrites of every osteocyte beside the blood vessel. (n = 119 osteocytes examined over 5 independent mice). h Quantitative percentage of dendrites in contact with vessels or in noncontact with vessels among all the vessel-nearside dendrites as described in g. i The relative angle between the long bone axis and the long axis of the osteocyte soma distant from TCVs (no dendrite contacts with TCVs) or near TCVs, as shown in d (142 osteocytes adjacent to TCVs and 368 osteocytes distant to TCVs were examined from independent 5 mice). Data were presented as the means ± SEMs; Significance was calculated using unpaired t test with two-tailed P value g, h, i. Source data are provided as a Source Data file.

Fig. 2. Osteocytes regulate TCVs and angiogenesis.

a Representative images of high-resolution µCT (1 µm resolution) on 6-week-old male mouse femurs presented the complicated canal network in DTA^ki/wt control mouse cortical bone and crude canal system in Dmp1^Cre-DTA^ki/wt mouse cortical bone. b Representative confocal images of CD31-labeled blood vessels and phalloidin-labeled osteocytes from 6-week-old male DTA^ki/wt and Dmp1^Cre-DTA^ki/wt mouse femur cortical bone. Scale bars, 100 µm. c, d Quantitative analysis of the number of osteocytes in contact with blood vessels c and the number of TCV branches d from 6-week-old male DTA^ki/wt and Dmp1^Cre-DTA^ki/wt mouse femur cortical bone as shown in b (Data were quantified from 5 biologically independent samples). e Top 10 downregulated GO terms in 4-week-old Dmp1^Cre-DTA^ki/wt mouse femur cortical bone compared with DTA^ki/wt control mice. f GSEA of angiogenesis (P < 0.0001, NES = −2.1858) in femur cortical bone of 4-week-old Dmp1^Cre-DTA^ki/wt mice compared with DTA^ki/wt control mice. g Selected significantly downregulated blood vessel-related GO terms in Dmp1^Cre-DTA^ki/wt mice. h Venn diagram of overlapping genes from reported endothelial cell-derived angiogenesis genes and downregulated genes related to blood vessels in Dmp1^Cre-DTA^ki/wt mice. i RT-qPCR analysis of the overlapping genes in h in cortical bone of 4-week-old DTA^ki/wt and Dmp1^Cre-DTA^ki/wt mouse femurs (n = 3 biologically independent samples). Data were presented as the means ± SEMs; Significance was calculated using unpaired t test with two-tailed P value c, d, i or hypergeometric test e, g or permutation test f. Source data are provided as a Source Data file.

Fig. 3. Mitochondrial transfer from osteocytes to endothelial cells.

a Flowchart of 2D coculture of MLO-Y4^Mito-Dendra2 and CellMask-labeled bEnd.3 endothelial cells. This figure was created by P.L. and D.L.L. and cartonized by Mr. Zihao Li. b Representative confocal images of CellMask-labeled bEnd.3 endothelial cells with orthogonal view show the acquisition of mitochondria derived from MLO-Y4^Mito-Dendra2 after coculture. Scale bars, 20 µm. c Schematic diagram for 2d-coculture and transwell coculture system of bEnd.3 cells and MLO-Y4 cells with number ratio at 3:1 (MLO-Y4: bEnd.3) for 48 hours. d, e Representative confocal images d and quantitative result e of bEnd.3 cells acquired with Mito-Dendra2 fluorescence as shown in c, scale bars, 50 µm, yellow arrows represent transferred mitochondria (n = 3 biologically independent samples). f Schematic diagram for the generation of the Dmp1^Cre-Cox8^Dendra2 mouse line. When crossed to the Dmp1^Cre mouse line, the termination cassette (STOP symbol) of Cox8^Dendra2 mice was removed to generate the Dmp1^Cre-Cox8^Dendra2 mouse line with osteocyte-specific labeling of mitochondria by Dendra2 green fluorescence. g Confocal images of femur cortical bone from a 4-week-old male Dmp1^Cre-Cox8^Dendra2 mouse show the translocation of osteocyte-derived mitochondria (Dendra2) in CD31-labeled endothelial cells. Scale bars, 20 µm. h Quantitative result of TCVs that acquired with Mito-Dendra2 fluorescence in femur cortical bone from 4-6-week-old Dmp1^Cre-Cox8a^Dendra2 (n = 4 biologically independent samples). i Schematic diagram for the generation of Dmp1^Cre-mGmT mice. When crossed to the Dmp1^Cre mouse line, the Loxp-flanked Zsgreen cassette will be removed to produce the Dmp1^Cre-mGmT mouse strain, in which the Dmp1⁺ cells will be labeled by tdTomato fluorescent protein while Dmp1^- cells will express Zsgreen fluorescence protein. j Confocal images of femur cortical bone collected from a 4-week-old male Dmp1^Cre-mGmT mouse revealed the adjacent but noncolocalized relationship between CD31-labeled endothelial cells and tdTomato-labeled Dmp1⁺ cells. Scale bars, 20 µm. Data were presented as the means ± SEMs. Significance was calculated using unpaired t test with two-tailed P value e. Source data are provided as a Source Data file.

Fig. 4. Impaired mitochondrial transfer by deleting Rhot1 in osteocytes leads to TCV regression.

a Confocal images show the decreased transfer of mitochondria from Rhot1^KD-MLO-Y4^Mito-Dendra2 cells to CD31-stained bEnd.3 cells after 24 h of coculture. Scale bars, 10 µm. b Workflow for the flow cytometry analysis of the mitochondrial acquisition efficiency in bEnd.3 cells after coculturing with NC-MLO-Y4^Mito-Dendra2 or Rhot1^KD-MLO-Y4 cells. This figure was created by P.L. and D.L.L. and cartonized by Mr. Zihao Li. c Representative dot-plots and quantitative result of the percentage of bEnd.3 cells acquired with Mito-Dendra2 fluorescence in entire bEnd.3 cells population after coculturing with NC-MLO-Y4^Mito-Dendra2 or Rhot1^KD-MLO-Y4 cells (n = 3 biologically independent samples). d Schematic diagram for the generation of the Dmp1^Cre-Cox8^Dendra2-Rhot1^fl/fl mouse line. e Representative confocal images of Dendra2-labeled mitochondria and CD31-labeled endothelial cells from the femur cortical bone of 6-week-old male Dmp1^Cre-Cox8^Dendra2 control mice and Dmp1^Cre-Cox8^Dendra2-Rhot1^fl/fl mice. Scale bars, 20 µm. f Quantitative assessment of the number of Dendra2-labeled mitochondria in each TCV as shown in e. (Data were quantified from three mice, and four different views per mouse were captured for the quantification). g Strategy diagram of the generation of the Dmp1^Cre-Rhot1^fl/fl mouse line for the specific deletion of the Rhot1 gene in Dmp1-expressing cells by crossing the Loxp-flanked Rhot1 allele with the Dmp1^Cre mouse line. h Representative confocal images of CD31-labeled blood vesselsfrom femur cortical bone of 6-week-old male Rhot1^fl/fl control mice or Dmp1^Cre-Rhot1^fl/fl mice. Scale bars, 200 µm. i Quantitative assessment of the number of TCV branches (white arrowhead) shown in h (Data were quantified from three mice, and upper and lower cortical bone of each mouse were captured for the quantification). j Representative images of high-resolution µCT (1 µm resolution) on 6-week-old male mouse femurs presenting the regressed canal network in Dmp1^Cre-Rhot1^fl/fl mice compared to Rhot1^fl/fl control mice. Data were presented as the means ± SEMs. Significance was calculated using unpaired t test with two-tailed P value c, f, i. Source data are provided as a Source Data file.

Fig. 5. Mitochondria transferred from osteocytes restore endothelial dysfunction.

a Confocal images of the acquisition of MLO-Y4-derived mitochondria (Mito) in bEnd.3 endothelial cells. b, c Extracellular flux analysis b quantitative results c and of OXPHOS activity in vehicle-treated healthy bEnd.3 cells, healthy bEnd.3 cells transplanted with MLO-Y4 cells mitochondria, 2 µM A/R-damaged bEnd.3 cells, and 2 µM A/R-damaged bEnd.3 cells transplanted with MLO-Y4 cells mitochondria (n = 3 biologically independent samples). d, e Representative histogram plot d and quantitative result e of DCFH-DA fluorescence (ROS) mean intensity of vehicle-treated bEnd.3 cells, 2 µM A/R-damaged bEnd.3 cells and 2 µM A/R-damaged bEnd.3 cells transplanted with MLO-Y4 cells mitochondria (n = 4 biologically independent samples). f, g CCK-8 cell proliferation assay f on vehicle-treated healthy bEnd.3 cells, 2 µM A/R-damaged bEnd.3 cells, and 2 µM A/R-damaged bEnd.3 cells transplanted with MLO-Y4 cells mitochondria at a ratio of 3:1 or 30:1 over 7 days and statistical result g of the OD value on day 3 (n = 6 biologically independent samples). h, i Representative images h and quantitative result i of the tube formation assay on vehicle-treated healthy bEnd.3 cells, 2 µM A/R-damaged bEnd.3 cells, and 2 µM A/R-damaged bEnd.3 cells transplanted with MLO-Y4 cells mitochondria at a ratio of 3:1 or 30:1 (n = 3 biologically independent samples). j, k Representative images j and quantitative result k of the wound healing assay on vehicle-treated bEnd.3 cells, 2 µM A/R-damaged bEnd.3 cells, and 2 µM A/R-damaged bEnd.3 cells transplanted with MLO-Y4 cells mitochondria at a ratio of 3:1 or 30:1 (n = 3 biologically independent samples). l Workflow for bone defect surgery and mitochondrial injection. m, n Representative confocal images m of femur callus in bone defected area and quantitative results n (n = 3 biologically independent samples). o, p Representative micro-CT images o of femur defects 1 week after surgery and histomorphometric analysis p of the regenerated bone (n = 9 biologically independent samples). Data were presented as the means ± SEMs; Significance was calculated based on unpaired t test with two-tailed P value c, e, g, n, p or one-way ANOVA followed by Tukey’s post hoc test i, k. Source data are provided as a Source Data file.

Fig. 6. Osteocyte-derived mitochondria regulate endothelial function by inducing D-sphingosine.

a Volcano plot of untargeted metabolomic analysis of the differentially detected (P < 0.05, FC > 1.2 or FC < 0.833, VIP > 1) metabolites in bEnd.3 cells transplanted with MLO-Y4 cells mitochondria. b Schematic of screening potential metabolites related to mitochondria that promote angiogenesis. c, d Representative histogram plot c and quantitative result d of ROS level of D-sphingosine treated healthy and damaged bEnd.3 cells. (n = 5 biologically independent samples). e, f The CCK-8 cell proliferation assay e of D-sphingosine treated healthy and damaged bEnd.3 cells throughout 5 days and statistical result f of the OD value on day 3 (n = 6 biologically independent samples). g, h Representative images g of the tube formation assay and quantitative result h of D-sphingosine treated healthy and damaged bEnd.3 cells. (n = 5 biologically independent samples). i, j Representative images i and quantitative result j of the wound healing assay of D-sphingosine treated healthy and damaged bEnd.3 cells. (n = 3 biologically independent samples). k, l Workflow k and the quantitative result l of sphingosine-1-phosphate (S1P) concentration in bEnd.3 cells, both with and without the acquisition of MLO-Y4 cells mitochondria. (n = 7 biologically independent samples). m Workflow for tube formation and wound healing assays using bEnd.3 endothelial cells transplanted with mitochondria from osteocytes pre-treated with PF543 citrate. This figure was created by P.L. and cartonized by Ms. Lina Cao. n, o Tube formation assay on differentially treated bEnd.3 cells transplanted with mitochondria from PF-543 citrate-induced MLO-Y4 cells n and statistical result o. (n = 5 biologically independent samples). p, q Wound healing assay p and quantitative result q on healthy, damaged and damaged bEnd.3 cells transplanted with mitochondria from normal or PF-543 citrate-induced MLO-Y4 cells (n = 3 biologically independent samples). r Workflow for femoral defect surgery and D-sphingosine treatment. s Representative micro-CT images of femur defects 1 week after surgery. t Histomorphometric analysis of the regenerated bone. (n = 5 biologically independent samples). Data were presented as the means ± SEMs. Significance was calculated using unpaired t test with two-tailed P value a, j, n, q, s or one way ANOVA followed by Turkey’s post hoc test d, f, h. Source data are provided as a Source Data file.

Fig. 7. Schematic diagram of mitochondria transferring from osteocytes to transcortical endothelial cells for maintaining TCVs vascularization.

Mitochondria transferred from osteocytes promote angiogenesis of TCVs via activating the production of endothelial D-sphingosine, which is further catalyzed into sphingosine-1-phosphate by the SPHK1 of osteocyte-derived mitochondria. This figure was created by P.L. and D.L.L. and cartonized by Ms. Lina Cao.