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.

References

[1] Shehab D, Elgazzar AH, Collier BD. Heterotopic ossification. J. Nucl. Med, 2002, 43: 346-353

[2] Meyers C et al. Heterotopic ossification: a comprehensive review. JBMR Plus, 2019, 3: e10172

[3] Wang X et al. Inhibition of overactive TGF-beta attenuates progression of heterotopic ossification in mice. Nat. Commun., 2018, 9

[4] Agarwal S et al. Scleraxis-lineage cells contribute to ectopic bone formation in muscle and tendon. Stem Cells, 2017, 35: 705-710

[5] Bi Y et al. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat. Med., 2007, 13: 1219-1227

[6] Lee CH et al. Harnessing endogenous stem/progenitor cells for tendon regeneration. J. Clin. Invest, 2015, 125: 2690-2701

[7] Yin Z et al. Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality. Sci. Adv., 2016, 2: e1600874

[8] Harvey T, Flamenco S, Fan CM. A Tapp3⁺ Pdgfra⁺ tendon stem cell population contributes to regeneration and reveals a shared role for PDGF signalling in regeneration and fibrosis. Nat. Cell Biol., 2019, 21: 1490-1503

[9] Ito Y et al. The Mohawk homeobox gene is a critical regulator of tendon differentiation. Proc. Natl. Acad. Sci. USA, 2010, 107: 10538-10542

[10] Kimura W et al. Irxl1 mutant mice show reduced tendon differentiation and no patterning defects in musculoskeletal system development. Genesis, 2011, 49: 2-9

[11] Liu H et al. Mohawk promotes the tenogenesis of mesenchymal stem cells through activation of the TGFbeta signaling pathway. Stem Cells, 2015, 33: 443-455

[12] Liu W et al. The atypical homeodomain transcription factor Mohawk controls tendon morphogenesis. Mol. Cell Biol., 2010, 30: 4797-4807

[13] Suzuki H et al. Gene targeting of the transcription factor Mohawk in rats causes heterotopic ossification of Achilles tendon via failed tenogenesis. Proc. Natl. Acad. Sci. USA, 2016, 113: 7840-7845

[14] Liu H, Xu J, Jiang R. Mkx-deficient mice exhibit hedgehog signaling-dependent ectopic ossification in the achilles tendons. J. Bone Min. Res., 2019, 34: 557-569

[15] Dejana E. The role of wnt signaling in physiological and pathological angiogenesis. Circ. Res, 2010, 107: 943-952

[16] Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411–420 (2018).

[17] Noack S et al. Periostin secreted by mesenchymal stem cells supports tendon formation in an ectopic mouse model. Stem Cells Dev., 2014, 23: 1844-1857

[18] Van den Plas D, Merregaert J. Constitutive overexpression of the integral membrane protein Itm2A enhances myogenic differentiation of C2C12 cells. Cell Biol. Int, 2004, 28: 199-207

[19] Van den Plas D, Merregaert J. In vitro studies on Itm2a reveal its involvement in early stages of the chondrogenic differentiation pathway. Biol. Cell, 2004, 96: 463-470

[20] Boeuf S et al. Enhanced ITM2A expression inhibits chondrogenic differentiation of mesenchymal stem cells. Differentiation, 2009, 78: 108-115

[21] Wenstrup RJ et al. Regulation of collagen fibril nucleation and initial fibril assembly involves coordinate interactions with collagens V and XI in developing tendon. J. Biol. Chem., 2011, 286: 20455-20465

[22] Docheva D, Hunziker EB, Fassler R, Brandau O. Tenomodulin is necessary for tenocyte proliferation and tendon maturation. Mol. Cell Biol., 2005, 25: 699-705

[23] Gehwolf R et al. Pleiotropic roles of the matricellular protein Sparc in tendon maturation and ageing. Sci. Rep., 2016, 6

[24] Qiu X et al. Single-cell mRNA quantification and differential analysis with Census. Nat. Methods, 2017, 14: 309-315

[25] Zhang Q, Zhou D, Wang H, Tan J. Heterotopic ossification of tendon and ligament. J. Cell Mol. Med, 2020, 24: 5428-5437

[26] Hilberg F et al. BIBF 1120: triple angiokinase inhibitor with sustained receptor blockade and good antitumor efficacy. Cancer Res., 2008, 68: 4774-4782

[27] Huang AH, Lu HH, Schweitzer R. Molecular regulation of tendon cell fate during development. J. Orthop. Res, 2015, 33: 800-812

[28] Sugg KB et al. Postnatal tendon growth and remodeling require platelet-derived growth factor receptor signaling. Am. J. Physiol. Cell Physiol., 2018, 314: C389-C403

[29] Tan, G. K. et al. Tgfbeta signaling is critical for maintenance of the tendon cell fate. Elife 9, e52695 (2020).

[30] Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature, 2014, 507: 323-328

[31] Cocks M et al. Vascular patterning in human heterotopic ossification. Hum. Pathol., 2017, 63: 165-170

[32] Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling - in control of vascular function. Nat. Rev. Mol. Cell Biol., 2006, 7: 359-371

[33] Andrae J, Gallini R, Betsholtz C. Role of platelet-derived growth factors in physiology and medicine. Genes Dev., 2008, 22: 1276-1312

[34] Ornitz DM, Itoh N. The fibroblast growth factor signaling pathway. Wiley Interdiscip. Rev. Dev. Biol., 2015, 4: 215-266

[35] Huang da W, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res., 2009, 37: 1-13

[36] Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc., 2009, 4: 44-57

[37] Chen S et al. AfterQC: automatic filtering, trimming, error removing and quality control for fastq data. BMC Bioinforma., 2017, 18

[38] Patel RK, Jain M. NGS QC Toolkit: a toolkit for quality control of next generation sequencing data. PLoS One, 2012, 7: e30619

[39] Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 2014, 30: 923-930

[40] Satija R, Farrell JA, Gennert D, Schier AF, Regev A. Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol., 2015, 33: 495-502

[41] Zou Z et al. A single-cell transcriptomic atlas of human skin aging. Dev. Cell, 2021, 56: 383-397 e388

[42] Ho TC et al. PEDF-derived peptide promotes tendon regeneration through its mitogenic effect on tendon stem/progenitor cells. Stem Cell Res. Ther., 2019, 10: 2

Funding

Ministry of Science and Technology of the People’s Republic of China (Chinese Ministry of Science and Technology)(2017YFA0104902)

National Natural Science Foundation of China (National Science Foundation of China)(81330041)

Science and Technology Department of Zhejiang Province(2013TD11)