Single cell analysis reveals inhibition of angiogenesis attenuates the progression of heterotopic ossification in Mkx-/- mice

Abstract

Tendon heterotopic ossification (HO) is characterized by bone formation inside tendon tissue, which severely debilitates people in their daily life. Current therapies fail to promote functional tissue repair largely due to our limited understanding of HO pathogenesis. Here, we investigate the pathological mechanism and propose a potential treatment method for HO. Immunofluorescence assays showed that the Mohawk (MKX) expression level was decreased in human tendon HO tissue, coinciding with spontaneous HO and the upregulated expression of osteochondrogenic and angiogenic genes in the tendons of Mkx^-/- mice. Single-cell RNA sequencing analyses of wild-type and Mkx^-/- tendons identified three cell types and revealed the excessive activation of osteochondrogenic genes during the tenogenesis of Mkx^-/- tendon cells. Single-cell analysis revealed that the gene expression program of angiogenesis, which is strongly associated with bone formation, was activated in all cell types during HO. Moreover, inhibition of angiogenesis by the small-molecule inhibitor BIBF1120 attenuated bone formation and angiogenesis in the Achilles tendons of both Mkx mutant mice and a rat traumatic model of HO. These findings provide new insights into the cellular mechanisms of tendon HO and highlight the inhibition of angiogenesis with BIBF1120 as a potential treatment strategy for HO.

Fig. 1

Mkx deficiency causes tendon heterotopic ossification. a HE (left) and immunofluorescence staining of OCN (middle) and MKX (right) in the tendons of normal individuals and HO patients. Scale bar, 50 μm. b, c Micro-CT examination of Achilles tendons (b) and patellar tendons (c) from WT and Mkx^−/− mice. Scale bar, 1 mm. d, e Safranin O-Fast green staining of Achilles tendons (d) and patellar tendons (e) from WT and Mkx^−/− mice. Scale bar, 50 μm

Fig. 2

Transcriptomic analysis of WT and Mkx^−/− tendons. a Volcano plot of differentially expressed genes between WT and Mkx^−/− tendons. b Representative enriched GO terms corresponding to upregulated genes in Mkx^−/− tendons. c Representative enriched GO terms corresponding to downregulated genes in Mkx^−/− tendons. d Boxplots of the expression levels of genes related to angiogenesis (Ctgf, Mmp2, Pecam1 and Runx1) and osteochondrogenesis (Sox9, Runx2, Spp1 and Postn) in WT and Mkx^−/− tendons. *:p < 0.05

Fig. 3

Single-cell profiles of WT and Mkx^−/− tendons. a, b t-SNE plot of WT and Mkx^−/− tendon cell clusters inferred from scRNA-seq data. The plots were visualized using CC1-CC4 and are colored by cell type (a) or genotype (b). CC: canonical vectors. c Numbers of WT and Mkx^−/− cells in each cell cluster. d Heatmap showing the marker genes for each cell cluster (left) and representative GO terms corresponding to these marker genes (right). TPCs tendon progenitor cells, TBs tenoblasts, TCs tenocytes

Fig. 4

Pseudotime analysis revealed shifted gene expression programs in Mkx^−/− tendon cells. a, b Order of the WT (a) and Mkx^−/− (b) cells along the differentiation trajectory based on pseudotime values and cell clusters. c, d Gene expression dynamics along the WT (c) and Mkx^−/− (d) differentiation trajectories. Genes (row) are clustered and cells (column) are ordered according to pseudotime development. e, f Kinetic diagrams showing the expression of early, middle and late tenogenesis markers during the differentiation of WT (e) and Mkx^−/− (f) cells. g, h Kinetic diagrams showing the expression of osteochondrogenic markers during the differentiation of WT (g) and Mkx^−/− (h) cells. i Violin plots showing the Spp1, Ogn, Mgp, and Comp expression in wild-type and Mkx^−/− TPCs, TBs, and TCs. ns no significance; *P < 0.05; **P < 0.01; ****P < 0.000 1. TPCs tendon progenitor cells, TBs tenoblasts, TCs tenocytes

Fig. 5

Differential gene expression analysis revealed elevated angiogenic gene expression in Mkx^−/− tendon cells. a–c Heatmap of the normalized gene expression of DEGs between WT and Mkx^−/− TPC (a), TB (b) and TC (c) clusters. d–f Representative GO terms corresponding to upregulated genes in the Mkx^−/− TPC (d), TB (e) and TC (f) clusters. g Violin plots of proangiogenic genes, including Hif1a, Thbs1, Vegfa, and Flt1, in the TPC, TB and TC clusters. h Safranin O-Fast green staining of the patellar tendons from WT and Mkx^−/− mice. The yellow arrows indicate the blood vessels. Scale bar, 50 μm. i Immunostaining of CD31 in WT and Mkx^−/− patellar tendons. Scale bar, 50 μm. ns no significance; *P < 0.05; **P < 0.01; ***P < 0.001. TPCs tendon progenitor cells, TBs tenoblasts, TCs tenocytes

Fig. 6

The angiogenesis inhibitor BIBF1120 attenuates heterotopic bone formation in the Mkx^−/− degenerative model of HO. a Schematic illustration of the treatment and sample collection workflow. b–e Micro-CT images of the Achilles tendons of 8-week-old (b) and 12-week-old (d) Mkx^−/− mice treated with BIBF1120 or vehicle. Scale bar, 1 mm. Quantitative analysis of the heterotopic bone volumes of the Achilles tendons of 8-week-old (c) and 12-week-old (e) Mkx^−/− mice. f Safranin O-Fast green staining of the Achilles tendons of normal, 8-week-old and 12-week-old Mkx^−/− mice. Scale bar, 50 μm. g Immunostaining of CD31⁺ (red) vessels. Scale bar, 50 μm. h Immunostaining of Ocn⁺ (red) cells in ectopic bone marrow. Scale bar, 25 μm. i, j Quantification of CD31⁺ (i) vessels and Ocn⁺ (j) cells. All data are shown as the mean ± s.d. n = 6 per group. Statistical test: student t test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 7

The angiogenesis inhibitor BIBF1120 attenuates heterotopic bone formation in a trauma-induced model of rat HO. a Schematic illustration of the treatment and sample collection workflow for the rat HO model. b–e Micro-CT images of the Achilles tendons of rats in the BIBF1120 and vehicle treatment groups at 9 (b) and 12 weeks (d) post ATP. Scale bar, 1 mm. Quantitative analysis of the heterotopic bone volumes of the Achilles tendons at 9 (c) and 12 weeks (e) postinjury. f Safranin O-Fast green staining of the rat Achilles tendons of normal rats at 9 and 12 weeks postinjury. Scale bar, 50 μm. g Immunostaining of CD31⁺ (red) vessels. Scale bar, 50 μm. h Immunostaining of Ocn⁺ (red) cells in ectopic bone marrow. Scale bar, 25 μm. i, j Quantification of CD31⁺ (i) vessels and Ocn⁺ (j) cells. All data are shown as the mean ± s.d. n = 8 per group. Statistic test: student t test. **P < 0.01; ***P < 0.001; ****P < 0.000 1