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. 2018 Feb 7;9(1):551.
doi: 10.1038/s41467-018-02988-5.

Inhibition of Overactive TGF-β Attenuates Progression of Heterotopic Ossification in Mice

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Free PMC article

Inhibition of Overactive TGF-β Attenuates Progression of Heterotopic Ossification in Mice

Xiao Wang et al. Nat Commun. .
Free PMC article

Abstract

Acquired heterotopic ossification (HO) is a painful and debilitating disease characterized by extraskeletal bone formation after injury. The exact pathogenesis of HO remains unknown. Here we show that TGF-β initiates and promotes HO in mice. We find that calcified cartilage and newly formed bone resorb osteoclasts after onset of HO, which leads to high levels of active TGF-β that recruit mesenchymal stromal/progenitor cells (MSPCs) in the HO microenvironment. Transgenic expression of active TGF-β in tendon induces spontaneous HO, whereas systemic injection of a TGF-β neutralizing antibody attenuates ectopic bone formation in traumatic and BMP-induced mouse HO models, and in a fibrodysplasia ossificans progressive mouse model. Moreover, inducible knockout of the TGF-β type II receptor in MSPCs inhibits HO progression in HO mouse models. Our study points toward elevated levels of active TGF-β as inducers and promoters of ectopic bone formation, and suggest that TGF-β might be a therapeutic target in HO.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Elevated active TGF-β levels in human HO. a H&E staining of normal muscle (left) and HO in early osteogenesis stage (middle) and late osteogenesis stage (right) after elbow fracture (EF) and central nervous system trauma (CNST). Scale bar, 100 μm. b Safranin O and Fast Green (SOFG) staining of normal muscle (left) and HO. Proteoglycan (red) and heterotopic bone (green). Scale bar, 100 μm. c TRAP+ cells (red) and d quantitative analysis of the number of TRAP+ osteoclast surface (OCS) per bone surface (BS). Scale bar, 100 μm. Immunohistochemical staining of e pSmad2/3+ cells (brown) 
and g PDGF-BB+ cells (brown) and quantitative analysis of the number of f pSmad2/3+ cells or h PDGF-BB+ cells per bone marrow area (mm2). i, j Quantitative analysis of i active TGF-β and j PDGF-BB in serum in HO patients at early osteogenesis stage and late mature stage by ELISA. Dotted line indicates the average concentration of healthy people with active TGF-β of 1.23 ng/ml and PDGF-BB of 1.68 ng/ml. k Immunofluorescent staining and l quantitative analysis of CD73+ (green) and CD90+ cells (red) in bone marrow of ectopic bone marrow in HO patients. Blue color indicates DAPI staining of nuclei. Scale bar, 20 μm. C cartilage, B bone, BM bone marrow, M muscle, NM normal muscle, EF elbow fracture, CNST central nervous system trauma, OR osteogenesis MR maturation. All data are shown as the mean ± s.d. n = 9 per group for d, f, h, l histomorphometry analysis. *p < 0.05 as determined by unpaired, two-tailed Student’s t-test. n = 8 per group for i, j ELISA analysis. *p< 0.05 as determined by paired, two-tailed Student’s t-test
Fig. 2
Fig. 2
Elevated active TGF-β levels were associated with increased angiogenesis in HO mice. a Micro CT images of Achilles tendon (sagittal view) after sham operation or at 3, 6, 9, and 15 weeks after ATP and b quantitative analysis. Scale bar, 2 mm. c TRAP staining (magenta) and d quantification of heterotopic bone in mouse Achilles tendon. e, g Immunohistochemical staining and f, h quantification of e, f pSmad2/3+ cells and g, h PDGF-BB+ cells after sham operation or ATP. Scale bar, 50 μm. i, j Quantitative analysis of i active TGF-β and j PDGF-BB in serum determined by ELISA. k Nestin+ (red) cells in the ectopic bone marrow of sham or ATP mice and l quantification. Scale bar, 50 μm. Blue indicates DAPI staining of nuclei. m Emcn+ (green) and CD31+ (red) cells and n quantification of the fold change of type H vessels in ATP mice normalized to that of sham mice (set to 1) in the ectopic bone marrow. Scale bar, 100 μm. Yellow indicates type H vessels. o Immunohistochemical staining and p quantification of Ocn+ cells after sham operation or ATP. All data are shown as the mean ± s.d. n = 8 per group. *p< 0.05 as determined by one-way ANOVA
Fig. 3
Fig. 3
Macrophages produce TGF-β to initiate HO. ah High levels of active TGF-β were associated with increased macrophage four days after HO induction by ATP. a, c Immunostaining and b, d quantitative analysis of CD68 and pSmad2/3-positive cells in Achilles tendon two days after sham operation or ATP. Scale bar, 50 μm. n = 8 per group. e Micro CT images and f quantitative analysis of HO bone volume in the Achilles tendons (sagittal view) in WT or CSF1−/− mice 6 weeks after ATP. Scale bar, 2 mm. n = 8 per group g Micro CT images and h quantification of HO bone volume in the Achilles tendons (sagittal view) in Tgfb1flox/flox or LysM-cre::Tgfb1flox/flox mice 6 weeks after ATP. Scale bar, 2 mm. n = 8 per group. eq Transgenic expression of active TGF-β by Col I promoter induces HO. i Micro CT images of the Achilles tendon (sagittal view) of 4-month-old CED mice and WT littermates and j quantitative analysis of HO bone volume in Achilles tendon. Scale bar, 2 mm k H&E staining of Achilles tendon of 4-month-old CED mice and WT littermates. Scale bar, 50 μm. l Nestin+ (red) cells and m quantification in the ectopic bone marrow of CED and WT mice. Scale bar, 50 μm. Blue indicates DAPI staining of nuclei. n CD31+ (red) and Emcn+ (green) cells in the ectopic bone marrow. Scale bar, 100 μm. Yellow indicates type H vessels. o Quantification of the fold change of type H vessels in CED mice compared to that of WT littermates. p Immunostaining and q quantification of Ocn+ cells of in ectopic bone marrow in Achilles tendons of 4-month-old CED mice and WT littermates. Scale bar, 20 μm. All data are shown as the mean ± s.d. n = 22 per group. *p < 0.05 as determined by unpaired Student’s t-test
Fig. 4
Fig. 4
Systemic injection of TGF-β neutralizing antibody reduces ectopic bone formation and type H vessel formation. ac Mice were treated with 5 mg/kg body weight of the TGF-β neutralizing antibody 1D11 three times a week for 3 weeks from 1 day (D1), 3 weeks (W3), 6 weeks (W6), and 12 weeks (W12) after ATP and analyzed 15 weeks after ATP or sham surgery. a Micro CT images of the Achilles tendon (sagittal view) of mice and b quantitative analysis of bone volume of heterotopic bone determined by μCT analysis. Scar bar, 2 mm. c H&E staining of ectopic bone of Achilles tendons. Scale bar, 50 μm. dl Mice were treated with 5 mg/kg body weight of 1D11 three times a week for 3 weeks from 1 day (D1), 3 weeks (W3), and 6 weeks (W6) after ATP and analyzed 9 weeks after ATP or sham surgery. d Immunostaining and e quantification of pSmad2/3+ cells in ectopic bone marrow. Scale bar, 50 μm. f Active TGF-β in serum determined by ELISA. g Nestin+ (red) cells in the ectopic bone marrow and h quantification. Scale bar, 50 μm. Blue indicates DAPI staining of nuclei. i CD31+ (red) and Emcn+ (green) cells in the ectopic bone marrow. j Quantification of the fold change of Type H vessels in 1D11-treated ATP mice normalized to that of Sham mice. Scale bar, 100 μm. Yellow indicates type H vessels. k Immunostaining and l quantification of Ocn+ cells of in ectopic bone marrow. Red arrow shows Ocn+ cells. Scale bar, 50 μm. All data are shown as the mean ± s.d. n = 8 per group. *p < 0.05 as determined by one-way ANOVA
Fig. 5
Fig. 5
TGF-β neutralizing antibody attenuates HO induced by BMP-2/gelatin implantation and FOP mice. ac Mice were treated with 5 mg/kg body weight of the TGF-β neutralizing antibody 1D11 three times a week for 2 weeks from 1 day (D1), 2 weeks (W2), and 4 weeks (W4) after BGI and analyzed 8 weeks after BGI or sham surgery. a micro CT images and b quantitative analysis of bone volume of heterotopic bone in hamstring muscles of mice. Scar bar, 4 mm. c H&E staining of ectopic bone in hamstring muscles. Scale bar, 100 μm. dk Mice were treated with 5 mg/kg body weight of 1D11 three times a week for 2 weeks from 1 day (D1) and 2 weeks (W2) after BGI and analyzed 4 weeks after BGI or sham surgery. d Immunostaining and e quantification of pSmad2/3+ cells in ectopic bone marrow. Scale bar, 50 μm. f Nestin+ (red) cells in the ectopic bone marrow and g quantification. Scale bar, 50 μm. Blue indicates DAPI staining of nuclei. h CD31+ (red) and Emcn+ (green) cells in the ectopic bone marrow and i quantification of the fold change of type H vessels normalized to that of Sham mice. Scale bar, 100 μm. Yellow indicates type H vessels. j Immunostaining and k quantification of Ocn+ cells of in ectopic bone marrow. Scale bar, 50 μm. l, m Ad.Cre and cobra venom factor-injected caALK2 transgenic mice were treated with vehicle or 1D11 three times a week for 3 weeks. l Micro CT images of hamstring muscles after vehicle or 1D11 treatment and m quantitative analysis of bone volume. Scale bar, 4 mm. All data are shown as the mean ± s.d. n = 8 per group. b, e, g, i, k *p < 0.05 as determined by one-way ANOVA. m *p < 0.05 as determined by unpaired, two-tailed Student’s t-test
Fig. 6
Fig. 6
Nestin+ cells at the injured site are predominantly MSPCs. a The percentage of the CD45-GFP+ cells that express LepR. n = 16. b The percentages of the GFP+LepR+ cells or c GFP+LepR cells that express Sca-1, CD105, or CD31, respectively. d Alizarin red staining of GFP+ cells cultured in osteogenic differentiation medium for 14 days and e tube formation of GFP+ cells by Matrigel assay. f Immunostaining of COLII+ cells (red) and YFP+ cells (green) and g quantitation of Nestin-creERT2::EYFPflox/flox mice 3 weeks post ATP. Scale bar, 50 μm, n = 8 per group. h, left: CD31+ cells (red), YFP+ cells (green), and right: Emcn+ cells (red), YFP+ cells (green), and i quantitation in ectopic bone marrow of Nestin-creERT2::EYFPflox/flox mice 6 weeks post ATP. Scale bar, 100 μm, n = 8 per group. All data are shown as the mean ± s.d
Fig. 7
Fig. 7
Inducible knockout of Tgfbr2 in Nestin+ cells attenuates HO formation in ATP and BGI mice. a, c Micro CT images and b, d quantifications of a, b Achilles tendons or c, d hamstring muscles of Nestin-creERT2::Tgfbr2flox/flox mice 2 months treated with vehicle or tamoxifen after undergoing ATP or BGI surgery, respectively. Scale bar, 2 mm. e, f SOFG staining of e Achilles tendons or f hamstring muscles of Tgfbr2flox/flox and Tgfbr2−/− mice 2 months after ATP or BGI, respectively. Scale bar, 50 μm g, i Nestin+ (red) cells in the ectopic bone marrow and h, j quantifications of Tgfbr2flox/flox and Tgfbr2−/− mice after ATP or BGI, respectively. Scale bar, 50 μm. Blue indicates DAPI staining of nuclei. k, m CD31+ (red) and Emcn+ (green) cells in the ectopic bone marrow and quantifications of Tgfbr2flox/flox and Tgfbr2−/− mice after ATP or BGI. Scale bars, 100 μm. Yellow indicates type H vessels. an n = 8 per group. All data are shown as the mean ± s.d. *p < 0.05 as determined by two-tailed, unpaired Student’s t-test. o Hypothetical diagram of HO formation. After HO induction, abundant immune cells are infiltrated in the injury sites and produce TGF-β. Next, the chondrocytes proliferate and undergo hypertrophy and calcification. Finally, osteoclasts resorb calcified cartilage and free TGF-β from latent protein and diffuse to the ectopic bone marrow cavity. Active TGF-β recruits MSPCs to form type H vessels and for osteoblastic differentiation

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