Neuron-to-vessel signaling is a required feature of aberrant stem cell commitment after soft tissue trauma

Abstract

The functional interdependence of nerves and blood vessels is a well-established concept during tissue morphogenesis, yet the role of neurovascular coupling in proper and aberrant tissue repair is an emerging field of interest. Here, we sought to define the regulatory relationship of peripheral nerves on vasculature in a severe extremity trauma model in mice, which results in aberrant cell fate and heterotopic ossification (HO). First, a high spatial degree of neurovascular congruency was observed to exist within extremity injury associated heterotopic ossification. Vascular and perivascular cells demonstrate characteristic responses to injury, as assessed by single cell RNA sequencing. This vascular response to injury was blunted in neurectomized mice, including a decrease in endothelial proliferation and type H vessel formation, and a downregulation of key transcriptional networks associated with angiogenesis. Independent mechanisms to chemically or genetically inhibit axonal ingrowth led to similar deficits in HO site angiogenesis, a reduction in type H vessels, and heterotopic bone formation. Finally, a combination of single cell transcriptomic approaches within the dorsal root ganglia identified key neural-derived angiogenic paracrine factors that may mediate neuron-to-vascular signaling in HO. These data provide further understanding of nerve-to-vessel crosstalk in traumatized soft tissues, which may reflect a key determinant of mesenchymal progenitor cell fate after injury.

References

[1] Mukouyama YS, Shin D, Britsch S, Taniguchi M, Anderson DJ. Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin. Cell, 2002, 109: 693-705

[2] Liu L, Dana R, Yin J. Sensory neurons directly promote angiogenesis in response to inflammation via substance P signaling. FASEB J., 2020, 34: 6229-6243

[3] Carmeliet P, Tessier-Lavigne M. Common mechanisms of nerve and blood vessel wiring. Nature, 2005, 436: 193-200

[4] Meyers CA et al. A neurotrophic mechanism directs sensory nerve transit in cranial bone. Cell Rep., 2020, 31: 107696

[5] Li Z et al. Fracture repair requires TrkA signaling by skeletal sensory nerves. J. Clin. Investig., 2019, 129: 5137-5150

[6] Lee S et al. NGF-TrkA signaling dictates neural ingrowth and aberrant osteochondral differentiation after soft tissue trauma. Nat. Commun., 2021, 12

[7] Martin P, Lewis J. Origins of the neurovascular bundle: interactions between developing nerves and blood vessels in embryonic chick skin. Int J. Dev. Biol., 1989, 33: 379-387

[8] Li W et al. Peripheral nerve-derived CXCL12 and VEGF-A regulate the patterning of arterial vessel branching in developing limb skin. Dev. Cell, 2013, 24: 359-371

[9] Honig MG, Frase PA, Camilli SJ. The spatial relationships among cutaneous, muscle sensory and motoneuron axons during development of the chick hindlimb. Development, 1998, 125: 995-1004

[10] Honig MG, Lance-Jones C, Landmesser L. The development of sensory projection patterns in embryonic chick hindlimb under experimental conditions. Dev. Biol., 1986, 118: 532-548

[11] Bates D, Taylor GI, Newgreen DF. The pattern of neurovascular development in the forelimb of the quail embryo. Dev. Biol., 2002, 249: 300-320

[12] Dorfman, H. D. & Czerniak, B. Bone Tumors, (Mosby, 1998).

[13] Bedi A et al. The incidence of heterotopic ossification after hip arthroscopy. Am. J. Sports Med., 2012, 40: 854-863

[14] Daniels CM et al. Has the proportion of combat-related amputations that develop heterotopic ossification increased? J. Orthop. Trauma, 2018, 32: 283-287

[15] Sandeep KN, Suresh G, Gopisankar B, Abhishek N, Sujiv A. Does excision of heterotopic ossification of the elbow result in satisfactory patient-rated outcomes? Malays. Orthop. J., 2017, 11: 35-40

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

[17] Ranganathan K et al. Heterotopic ossification: basic-science principles and clinical correlates. J. Bone Jt. Surg. Am., 2015, 97: 1101-1111

[18] Hoch B, Montag A. Reactive bone lesions mimicking neoplasms. Semin. Diagn. Pathol., 2011, 28: 102-112

[19] Rosenberg AE. Pseudosarcomas of soft tissue. Arch. Pathol. Lab. Med., 2008, 132: 579-586

[20] Reznik JE et al. A preliminary investigation on the effect of extracorporeal shock wave therapy as a treatment for neurogenic heterotopic ossification following traumatic brain injury. Part I: Eff. Pain. Brain Inj., 2017, 31: 526-532

[21] Tran L, Stein N, Miller S. Fibrodysplasia ossificans progressiva: early diagnosis is critical yet challenging. J. Pediatr., 2010, 157: 860.e1

[22] Kaplan FS et al. Fibrodysplasia ossificans progressiva. Best. Pr. Res. Clin. Rheumatol., 2008, 22: 191-205

[23] Kaplan FS et al. Early diagnosis of fibrodysplasia ossificans progressiva. Pediatrics, 2008, 121: e1295-e1300

[24] Salisbury E et al. Sensory nerve induced inflammation contributes to heterotopic ossification. J. Cell Biochem., 2011, 112: 2748-2758

[25] Salisbury E, Sonnet C, Heggeness M, Davis AR, Olmsted-Davis E. Heterotopic ossification has some nerve. Crit. Rev. Eukar Gene, 2010, 20: 313-324

[26] Abeynayake N, Arthur A, Gronthos S. Crosstalk between skeletal and neural tissues is critical for skeletal health. Bone, 2021, 142: 115645

[27] Olmsted-Davis EA et al. Progenitors in peripheral nerves launch heterotopic ossification. Stem Cells Transl. Med., 2017, 6: 1109-1119

[28] Chen M et al. Skeleton-vasculature chain reaction: a novel insight into the mystery of homeostasis. Bone Res, 2021, 9: 21

[29] Hwang C et al. Mesenchymal VEGFA induces aberrant differentiation in heterotopic ossification. Bone Res., 2019, 7: 36

[30] Agarwal S et al. Inhibition of Hif1 alpha prevents both trauma-induced and genetic heterotopic ossification. P Natl Acad. Sci. USA, 2016, 113: E338-E347

[31] Agarwal S et al. Analysis of bone-cartilage-stromal progenitor populations in trauma induced and genetic models of heterotopic ossification. Stem Cells, 2016, 34: 1692-1701

[32] Agarwal S et al. Surgical excision of heterotopic ossification leads to re-emergence of mesenchymal stem cell populations responsible for recurrence. Stem Cells Transl. Med., 2017, 6: 799-806

[33] Peterson JR et al. Direct mouse trauma/burn model of heterotopic ossification. J. Vis. Exp, 2015, 102: e52880

[34] Blanco R, Gerhardt H. VEGF and Notch in tip and stalk cell selection. Cold Spring Harb. Perspect. Med., 2013, 3: a006569

[35] Peng Y, Wu S, Li Y, Crane JL. Type H blood vessels in bone modeling and remodeling. Theranostics, 2020, 10: 426-436

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

[37] Renthal W et al. Transcriptional reprogramming of distinct peripheral sensory neuron subtypes after axonal injury. Neuron, 2020, 108: 128-144.e9

[38] Nyberg P, Xie L, Kalluri R. Endogenous inhibitors of angiogenesis. Cancer Res., 2005, 65: 3967-3979

[39] Behr B et al. Fgf-18 is required for osteogenesis but not angiogenesis during long bone repair. Tissue Eng. Part A, 2011, 17: 2061-2069

[40] Orlandini M et al. Vascular endothelial growth factor-D activates VEGFR-3 expressed in osteoblasts inducing their differentiation. J. Biol. Chem., 2006, 281: 17961-17967

[41] Dai J, Rabie ABM. VEGF: an essential mediator of both angiogenesis and endochondral ossification. J. Dent. Res., 2007, 86: 937-950

[42] Duan X et al. Vegfa regulates perichondrial vascularity and osteoblast differentiation in bone development. Development, 2015, 142: 1984-1991

[43] Yang YQ et al. The role of vascular endothelial growth factor in ossification. Int J. Oral. Sci., 2012, 4: 64-68

[44] Hu K, Olsen BR. Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair. J. Clin. Investig., 2016, 126: 509-526

[45] He FL, Soriano P. Dysregulated PDGFR alpha signaling alters coronal suture morphogenesis and leads to craniosynostosis through endochondral ossification. Development, 2017, 144: 4026-4036

[46] Ding H et al. A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling. Nat. Genet, 2004, 36: 1111-1116

[47] Hsieh HHS et al. Evaluation of salivary cytokines for diagnosis of both trauma-induced and genetic heterotopic ossification. Front. Endocrinol, 2017, 8: 74

[48] Ranganathan K et al. The role of the adaptive immune system in burn-induced heterotopic ossification and mesenchymal cell osteogenic differentiation. J. Surg. Res., 2016, 206: 53-61

[49] Kan LX et al. Dysregulation of local stem/progenitor cells as a common cellular mechanism for heterotopic ossification. Stem Cells, 2009, 27: 150-156

[50] Genet F et al. Neurological heterotopic ossification following spinal cord injury is triggered by macrophage-mediated inflammation in muscle. J. Pathol., 2015, 236: 229-240

[51] Sorkin M et al. Regulation of heterotopic ossification by monocytes in a mouse model of aberrant wound healing. Nat. Commun., 2020, 11

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

[53] Kraft CT et al. Trauma-induced heterotopic bone formation and the role of the immune system: A review. J. Trauma Acute Care Surg., 2016, 80: 156-165

[54] Chen Q et al. Fate decision of mesenchymal stem cells: adipocytes or osteoblasts? Cell Death Differ., 2016, 23: 1128-1139

[55] Cao RH, Farnebo J, Kurimoto M, Cao YH. Interleukin-18 acts as an angiogenesis and tumor suppressor. Faseb J., 1999, 13: 2195-2202

[56] Shen JK et al. Interleukin-18 has antipermeablity and antiangiogenic activities in the eye: reciprocal suppression with VEGF. J. Cell Physiol., 2014, 229: 974-983

[57] Dai SM, Nishioka K, Yudoh K. Interleukin (IL) 18 stimulates osteoclast formation through synovial T cells in rheumatoid arthritis: comparison with IL1 beta and tumour necrosis factor alpha. Ann. Rheum. Dis., 2004, 63: 1379-1386

[58] Zhu S et al. Chondromodulin-1 in health, osteoarthritis, cancer, and heart disease. Cell Mol. Life Sci., 2019, 76: 4493-4502

[59] Nakamichi Y et al. Chondromodulin I is a bone remodeling factor. Mol. Cell Biol., 2003, 23: 636-644

[60] Yukata K et al. Altered fracture callus formation in chondromodulin-I deficient mice. Bone, 2008, 43: 1047-1056

[61] Shukunami C, Hiraki Y. Role of cartilage-derived anti-angiogenic factor, chondromodulin-I, during endochondral bone formation. Osteoarthr. Cartil., 2001, 9: S91-S101

[62] Shore EM, Kaplan FS. Inherited human diseases of heterotopic bone formation. Nat. Rev. Rheumatol., 2010, 6: 518-527

[63] Tower RJ et al. Spatial transcriptomics reveals a role for sensory nerves in preserving cranial suture patency through modulation of BMP/TGF-beta signaling. Proc. Natl. Acad. Sci. USA, 2021, 118: e2103087118

[64] Chen X et al. A chemical-genetic approach to studying neurotrophin signaling. Neuron, 2005, 46: 13-21

[65] Tomlinson RE et al. NGF-TrkA signaling by sensory nerves coordinates the vascularization and ossification of developing endochondral bone. Cell Rep., 2016, 16: 2723-2735

[66] Ashraf S, Bouhana KS, Pheneger J, Andrews SW, Walsh DA. Selective inhibition of tropomyosin-receptor-kinase A (TrkA) reduces pain and joint damage in two rat models of inflammatory arthritis. Arthritis Res. Ther., 2016, 18: 97

[67] Nwosu LN, Mapp PI, Chapman V, Walsh DA. Blocking the tropomyosin receptor kinase A (TrkA) receptor inhibits pain behaviour in two rat models of osteoarthritis. Ann. Rheum. Dis., 2016, 75: 1246-1254

Funding

NIH/NIAMS R01 AR070773 NIH/NIDCR R21 DE027922