ABSTRACT
The Neurofibromatosis Type 1 (NF1) RASopathy is associated with persistent fibrotic nonunions (pseudarthrosis) in human and mouse skeletal tissue. Here, we first performed spatial transcriptomics to define the molecular signatures across normal endochondral healing following fracture in mice. Within the control fracture callus, we observed spatially restricted activation of morphogenetic pathways, such as TGF-ß, WNT, and BMP. To investigate the molecular mechanisms contributing to Nf1-deficient delayed fracture healing, we performed spatial transcriptomic analysis on a Postn-cre;Nf1flox/- (Nf1Postn) fracture callus. Transcriptional analyses, subsequently confirmed through p-SMAD1/5/8 immunohistochemistry, demonstrated a lack of BMP pathway induction in Nf1Postn mice. To further inform the human disease, we performed spatial transcriptomic analysis of fracture pseudarthrosis tissue from a NF1 patient. Analyses detected increased MAPK signaling at the fibrocartilaginous-osseus junction. Similar to the Nf1Postn fracture, BMP pathway activation was absent within the pseudarthrosis tissue. Our results demonstrate the feasibility to delineate the molecular and tissue-specific heterogeneity inherent in complex regenerative processes, such as fracture healing, and to reconstruct phase transitions representing endochondral bone formation in vivo. Furthermore, our results provide in situ molecular evidence of impaired BMP signaling underlying NF1 pseudarthrosis, potentially informing the clinical relevance of off-label BMP2 as a therapeutic intervention.
ABSTRACT
Human blood vessel walls show concentric layers, with the outermost tunica adventitia harboring mesenchymal progenitor cells. These progenitor cells maintain vessel homeostasis and provide a robust cell source for cell-based therapies. However, human adventitial stem cell niche has not been studied in detail. Here, using spatial and single-cell transcriptomics, we characterized the phenotype, potential, and microanatomic distribution of human perivascular progenitors. Initially, spatial transcriptomics identified heterogeneity between perivascular layers of arteries and veins and delineated the tunica adventitia into inner and outer layers. From this spatial atlas, we inferred a hierarchy of mesenchymal progenitors dictated by a more primitive cell with a high surface expression of CD201 (PROCR). When isolated from humans and mice, CD201Low expression typified a mesodermal committed subset with higher osteogenesis and less proliferation than CD201High cells, with a downstream effect on canonical Wnt signaling through DACT2. CD201Low cells also displayed high translational potential for bone tissue generation.
ABSTRACT
The profound pain accompanying bone fracture is mediated by somatosensory neurons, which also appear to be required to initiate bone regeneration following fracture. Surprisingly, the precise neuroanatomical circuitry mediating skeletal nociception and regeneration remains incompletely understood. Here, we characterized somatosensory dorsal root ganglia (DRG) afferent neurons innervating murine long bones before and after experimental long bone fracture in mice. Retrograde labeling of DRG neurons by an adeno-associated virus with peripheral nerve tropism showed AAV-tdT signal. Single cell transcriptomic profiling of 6,648 DRG neurons showed highest labeling across CGRP+ neuron clusters (6.9-17.2%) belonging to unmyelinated C fibers, thinly myelinated Aδ fibers and Aß-Field LTMR (9.2%). Gene expression profiles of retrograde labeled DRG neurons over multiple timepoints following experimental stress fracture revealed dynamic changes in gene expression corresponding to the acute inflammatory ( S100a8 , S100a9 ) and mechanical force ( Piezo2 ). Reparative phase after fracture included morphogens such as Tgfb1, Fgf9 and Fgf18 . Two methods to surgically or genetically denervate fractured bones were used in combination with scRNA-seq to implicate defective mesenchymal cell proliferation and osteodifferentiation as underlying the poor bone repair capacity in the presence of attenuated innervation. Finally, multi-tissue scRNA-seq and interactome analyses implicated neuron-derived FGF9 as a potent regulator of fracture repair, a finding compatible with in vitro assessments of neuron-to-skeletal mesenchyme interactions.
ABSTRACT
The regeneration of the mammalian skeleton's craniofacial bones necessitates the action of intrinsic and extrinsic inductive factors from multiple cell types, which function hierarchically and temporally to control the differentiation of osteogenic progenitors. Single-cell transcriptomics of developing mouse calvarial suture recently identified a suture mesenchymal progenitor population with previously unappreciated tendon- or ligament-associated gene expression profile. Here, we developed a Mohawk homeobox (MkxCG; R26RtdT) reporter mouse and demonstrated that this reporter identifies an adult calvarial suture resident cell population that gives rise to calvarial osteoblasts and osteocytes during homeostatic conditions. Single-cell RNA sequencing (scRNA-Seq) data reveal that Mkx+ suture cells display a progenitor-like phenotype with expression of teno-ligamentous genes. Bone injury with Mkx+ cell ablation showed delayed bone healing. Remarkably, Mkx gene played a critical role as an osteo-inhibitory factor in calvarial suture cells, as knockdown or knockout resulted in increased osteogenic differentiation. Localized deletion of Mkx in vivo also resulted in robustly increased calvarial defect repair. We further showed that mechanical stretch dynamically regulates Mkx expression, in turn regulating calvarial cell osteogenesis. Together, we define Mkx+ cells within the suture mesenchyme as a progenitor population for adult craniofacial bone repair, and Mkx acts as a mechanoresponsive gene to prevent osteogenic differentiation within the stem cell niche.
Subject(s)
Cell Differentiation , Homeodomain Proteins , Osteogenesis , Skull , Animals , Mice , Homeodomain Proteins/metabolism , Homeodomain Proteins/genetics , Osteogenesis/genetics , Skull/metabolism , Osteoblasts/metabolism , Osteoblasts/cytology , Cranial Sutures/metabolism , Stem Cells/metabolism , Stem Cells/cytology , Biomarkers/metabolismABSTRACT
Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels.
Subject(s)
Lymphangiogenesis , Mesenchymal Stem Cells , Ossification, Heterotopic , Vascular Endothelial Growth Factor C , Animals , Ossification, Heterotopic/metabolism , Ossification, Heterotopic/pathology , Ossification, Heterotopic/genetics , Vascular Endothelial Growth Factor C/metabolism , Vascular Endothelial Growth Factor C/genetics , Mice , Mesenchymal Stem Cells/metabolism , Lymphatic Vessels/metabolism , Lymphatic Vessels/pathology , Cell Differentiation , Tenocytes/metabolism , Osteogenesis , Haploinsufficiency , Mice, Inbred C57BL , Disease Models, Animal , MaleABSTRACT
While hypoxic signaling has been shown to play a role in many cellular processes, its role in metabolism-linked extracellular matrix (ECM) organization and downstream processes of cell fate after musculoskeletal injury remains to be determined. Heterotopic ossification (HO) is a debilitating condition where abnormal bone formation occurs within extra-skeletal tissues. Hypoxia and hypoxia-inducible factor 1α (HIF-1α) activation have been shown to promote HO. However, the underlying molecular mechanisms by which the HIF-1α pathway in mesenchymal progenitor cells (MPCs) contributes to pathologic bone formation remain to be elucidated. Here, we used a proven mouse injury-induced HO model to investigate the role of HIF-1α on aberrant cell fate. Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics analyses of the HO site, we found that collagen ECM organization is the most highly up-regulated biological process in MPCs. Zeugopod mesenchymal cell-specific deletion of Hif1α (Hoxa11-CreERT2; Hif1afl/fl) significantly mitigated HO in vivo. ScRNA-seq analysis of these Hoxa11-CreERT2; Hif1afl/fl mice identified the PLOD2/LOX pathway for collagen cross-linking as downstream of the HIF-1α regulation of HO. Importantly, our scRNA-seq data and mechanistic studies further uncovered that glucose metabolism in MPCs is most highly impacted by HIF-1α deletion. From a translational aspect, a pan-LOX inhibitor significantly decreased HO. A newly screened compound revealed that the inhibition of PLOD2 activity in MPCs significantly decreased osteogenic differentiation and glycolytic metabolism. This suggests that the HIF-1α/PLOD2/LOX axis linked to metabolism regulates HO-forming MPC fate. These results suggest that the HIF-1α/PLOD2/LOX pathway represents a promising strategy to mitigate HO formation.
Subject(s)
Ossification, Heterotopic , Osteogenesis , Animals , Mice , Collagen/metabolism , Disease Models, Animal , Extracellular Matrix/genetics , Hypoxia/metabolism , Ossification, Heterotopic/metabolism , Transcription Factors/metabolismABSTRACT
Intense inflammatory response impairs bone marrow mesenchymal stem cell (BMSC)-mediated bone regeneration, with transforming growth factor (TGF)-ß1 being the most highly expressed cytokine. However, how to find effective and safe means to improve bone formation impaired by excessive TGF-ß1 remains unclear. In this study, we found that the expression of orphan nuclear receptor Nr4a1, an endogenous repressor of TGF-ß1, was suppressed directly by TGF-ß1-induced Smad3 and indirectly by Hdac4, respectively. Importantly, Nr4a1 overexpression promoted BMSC osteogenesis and reversed TGF-ß1-mediated osteogenic inhibition and pro-fibrotic effects. Transcriptomic and histologic analyses confirmed that upregulation of Nr4a1 increased the transcription of Wnt family member 4 (Wnt4) and activated Wnt pathway. Mechanistically, Nr4a1 bound to the promoter of Wnt4 and regulated its expression, thereby enhancing the osteogenic capacity of BMSCs. Moreover, treatment with Nr4a1 gene therapy or Nr4a1 agonist Csn-B could promote ectopic bone formation, defect repair, and fracture healing. Finally, we demonstrated the correlation of NR4A1 with osteogenesis and the activation of the WNT4/ß-catenin pathway in human BMSCs and fracture samples. Taken together, these findings uncover the critical role of Nr4a1 in bone formation and alleviation of inflammation-induced bone regeneration disorders, and suggest that Nr4a1 has the potential to be a therapeutic target for accelerating bone healing.
Subject(s)
Bone Regeneration , Inflammation , Mesenchymal Stem Cells , Nuclear Receptor Subfamily 4, Group A, Member 1 , Osteogenesis , Wnt4 Protein , Mesenchymal Stem Cells/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Osteogenesis/genetics , Bone Regeneration/genetics , Animals , Mice , Wnt4 Protein/metabolism , Wnt4 Protein/genetics , Humans , Inflammation/genetics , Inflammation/metabolism , Gene Expression Regulation , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta1/genetics , Wnt Signaling Pathway , Male , Transcription, Genetic , Histone Deacetylases/metabolism , Histone Deacetylases/genetics , Disease Models, AnimalABSTRACT
Peripheral neurons terminate at the surface of tendons partly to relay nociceptive pain signals; however, the role of peripheral nerves in tendon injury and repair remains unclear. Here, we show that after Achilles tendon injury in mice, there is new nerve growth near tendon cells that express nerve growth factor (NGF). Conditional deletion of the Ngf gene in either myeloid or mesenchymal mouse cells limited both innervation and tendon repair. Similarly, inhibition of the NGF receptor tropomyosin receptor kinase A (TrkA) abrogated tendon healing in mouse tendon injury. Sural nerve transection blocked the postinjury increase in tendon sensory innervation and the expansion of tendon sheath progenitor cells (TSPCs) expressing tubulin polymerization promoting protein family member 3. Single cell and spatial transcriptomics revealed that disruption of sensory innervation resulted in dysregulated inflammatory signaling and transforming growth factor-ß (TGFß) signaling in injured mouse tendon. Culture of mouse TSPCs with conditioned medium from dorsal root ganglia neuron further supported a role for neuronal mediators and TGFß signaling in TSPC proliferation. Transcriptomic and histologic analyses of injured human tendon biopsy samples supported a role for innervation and TGFß signaling in human tendon regeneration. Last, treating mice after tendon injury systemically with a small-molecule partial agonist of TrkA increased neurovascular response, TGFß signaling, TSPC expansion, and tendon tissue repair. Although further studies should investigate the potential effects of denervation on mechanical loading of tendon, our results suggest that peripheral innervation is critical for the regenerative response after acute tendon injury.
Subject(s)
Nerve Growth Factor , Tendon Injuries , Animals , Humans , Mice , Cell Proliferation , Nerve Growth Factor/metabolism , Nerve Growth Factor/pharmacology , Stem Cells , Tendons/metabolism , Transforming Growth Factor beta , Receptor, trkA/metabolismABSTRACT
Self-renewal and differentiation of skeletal stem and progenitor cells (SSPCs) are tightly regulated processes, with SSPC dysregulation leading to progressive bone disease. While the application of single-cell RNA sequencing (scRNAseq) to the bone field has led to major advancements in our understanding of SSPC heterogeneity, stem cells are tightly regulated by their neighboring cells which comprise the bone marrow niche. However, unbiased interrogation of these cells at the transcriptional level within their native niche environment has been challenging. Here, we combined spatial transcriptomics and scRNAseq using a predictive modeling pipeline derived from multiple deconvolution packages in adult mouse femurs to provide an endogenous, in vivo context of SSPCs within the niche. This combined approach localized SSPC subtypes to specific regions of the bone and identified cellular components and signaling networks utilized within the niche. Furthermore, the use of spatial transcriptomics allowed us to identify spatially restricted activation of metabolic and major morphogenetic signaling gradients derived from the vasculature and bone surfaces that establish microdomains within the marrow cavity. Overall, we demonstrate, for the first time, the feasibility of applying spatial transcriptomics to fully mineralized tissue and present a combined spatial and single-cell transcriptomic approach to define the cellular components of the stem cell niche, identify cellâcell communication, and ultimately gain a comprehensive understanding of local and global SSPC regulatory networks within calcified tissue.
Subject(s)
Bone Marrow , Transcriptome , Animals , Mice , Bone Marrow/metabolism , Transcriptome/genetics , Bone and Bones , Stem Cells/metabolism , Cell Differentiation/geneticsABSTRACT
Modulation of the immune response to initiate and halt the inflammatory process occurs both at the site of injury as well as systemically. Due to the evolving role of cellular metabolism in regulating cell fate and function, tendon injuries that undergo normal and aberrant repair were evaluated by metabolic profiling to determine its impact on healing outcomes. Metabolomics revealed an increasing abundance of the immunomodulatory metabolite itaconate within the injury site. Subsequent single-cell RNA-Seq and molecular and metabolomic validation identified a highly mature neutrophil subtype, not macrophages, as the primary producers of itaconate following trauma. These mature itaconate-producing neutrophils were highly inflammatory, producing cytokines that promote local injury fibrosis before cycling back to the bone marrow. In the bone marrow, itaconate was shown to alter hematopoiesis, skewing progenitor cells down myeloid lineages, thereby regulating systemic inflammation. Therapeutically, exogenous itaconate was found to reduce injury-site inflammation, promoting tenogenic differentiation and impairing aberrant vascularization with disease-ameliorating effects. These results present an intriguing role for cycling neutrophils as a sensor of inflammation induced by injury - potentially regulating immune cell production in the bone marrow through delivery of endogenously produced itaconate - and demonstrate a therapeutic potential for exogenous itaconate following tendon injury.
Subject(s)
Neutrophils , Succinates , Humans , Neutrophils/metabolism , Succinates/pharmacology , Succinates/metabolism , Succinates/therapeutic use , Macrophages/metabolism , Inflammation/metabolismABSTRACT
OBJECTIVE: To characterize the role of neutrophil extracellular traps (NETs) in heterotopic ossification (HO) formation and progression and to use mechanical and pharmacological methods to decrease NETosis and mitigate HO formation. BACKGROUND: Traumatic HO is the aberrant osteochondral differentiation of mesenchymal progenitor cells after traumatic injury, burns, or surgery. While the innate immune response has been shown to be necessary for HO formation, the specific immune cell phenotype and function remain unknown. Neutrophils, one of the earliest immune cells to respond after HO-inducing injuries, can extrude DNA, forming highly inflammatory NETs. We hypothesized that neutrophils and NETs would be diagnostic biomarkers and therapeutic targets for the detection and mitigation of HO. METHODS: C57BL6J mice underwent burn/tenotomy (a well-established mouse model of HO) or a non-HO-forming sham injury. These mice were either (1) ambulated ad libitum, (2) ambulated ad libitum with daily intraperitoneal hydroxychloroquine, ODN-2088 (both known to affect NETosis pathways), or control injections, or (3) had the injured hind limb immobilized. Single-cell analysis was performed to analyze neutrophils, NETosis, and downstream signaling after the HO-forming injury. Immunofluorescence microscopy was used to visualize NETosis at the HO site and neutrophils were identified using flow cytometry. Serum and cell lysates from HO sites were analyzed using enzyme-linked immunosorbent assay for myeloperoxidase-DNA and ELA2-DNA complexes to identify NETosis. Micro-computerized tomography was performed on all groups to analyze the HO volume. RESULTS: Molecular and transcriptional analyses revealed the presence of NETs within the HO injury site, which peaked in the early phases after injury. These NETs were highly restricted to the HO site, with gene signatures derived from both in vitro NET induction and clinical neutrophil characterizations showing a high degree of NET "priming" at the site of injury, but not in neutrophils in the blood or bone marrow. Cell-cell communication analyses revealed that this localized NET formation coincided with high levels of toll-like receptor signaling specific to neutrophils at the injury site. Reducing the overall neutrophil abundance within the injury site, either pharmacologically through treatment with hydroxychloroquine, the toll-like receptor 9 inhibitor OPN-2088, or mechanical treatment with limb offloading, results in the mitigation of HO formation. CONCLUSIONS: These data provide a further understanding of the ability of neutrophils to form NETs at the injury site, clarify the role of neutrophils in HO, and identify potential diagnostic and therapeutic targets for HO mitigation.
Subject(s)
Extracellular Traps , Neutrophils , Animals , Mice , Neutrophils/metabolism , Hydroxychloroquine/metabolism , Extracellular Traps/metabolism , Immunity, Innate , DNA/metabolismABSTRACT
OBJECTIVE: Our objective was to identify macrophage subpopulations and gene signatures associated with regenerative or fibrotic healing across different musculoskeletal injury types. BACKGROUND: Subpopulations of macrophages are hypothesized to fine tune the immune response after damage, promoting either normal regenerative, or aberrant fibrotic healing. METHODS: Mouse single-cell RNA sequencing data before and after injury were assembled from models of musculoskeletal injury, including regenerative and fibrotic mouse volumetric muscle loss (VML), regenerative digit tip amputation, and fibrotic heterotopic ossification. R packages Harmony , MacSpectrum , and Seurat were used for data integration, analysis, and visualizations. RESULTS: There was a substantial overlap between macrophages from the regenerative VML (2 mm injury) and regenerative bone models, as well as a separate overlap between the fibrotic VML (3 mm injury) and fibrotic bone (heterotopic ossification) models. We identified 2 fibrotic-like (FL 1 and FL 2) along with 3 regenerative-like (RL 1, RL 2, and RL 3) subpopulations of macrophages, each of which was transcriptionally distinct. We found that regenerative and fibrotic conditions had similar compositions of proinflammatory and anti-inflammatory macrophages, suggesting that macrophage polarization state did not correlate with healing outcomes. Receptor/ligand analysis of macrophage-to-mesenchymal progenitor cell crosstalk showed enhanced transforming growth factor ß in fibrotic conditions and enhanced platelet-derived growth factor signaling in regenerative conditions. CONCLUSION: Characterization of macrophage subtypes could be used to predict fibrotic responses following injury and provide a therapeutic target to tune the healing microenvironment towards more regenerative conditions.
Subject(s)
Muscle, Skeletal , Ossification, Heterotopic , Mice , Animals , Macrophages , Wound Healing/physiology , Platelet-Derived Growth FactorABSTRACT
Extracellular matrix (ECM) interactions regulate both the cell transcriptome and proteome, thereby determining cell fate. Traumatic heterotopic ossification (HO) is a disorder characterized by aberrant mesenchymal lineage (MLin) cell differentiation, forming bone within soft tissues of the musculoskeletal system following traumatic injury. Recent work has shown that HO is influenced by ECM-MLin cell receptor signaling, but how ECM binding affects cellular outcomes remains unclear. Using time course transcriptomic and proteomic analyses, we identified discoidin domain receptor 2 (DDR2), a cell surface receptor for fibrillar collagen, as a key MLin cell regulator in HO formation. Inhibition of DDR2 signaling, through either constitutive or conditional Ddr2 deletion or pharmaceutical inhibition, reduced HO formation in mice. Mechanistically, DDR2 perturbation alters focal adhesion orientation and subsequent matrix organization, modulating Focal Adhesion Kinase (FAK) and Yes1 Associated Transcriptional Regulator and WW Domain Containing Transcription Regulator 1 (YAP/TAZ)-mediated MLin cell signaling. Hence, ECM-DDR2 interactions are critical in driving HO and could serve as a previously unknown therapeutic target for treating this disease process.
Subject(s)
Discoidin Domain Receptor 2 , Mice , Animals , Discoidin Domain Receptor 2/genetics , Proteomics , Cell Differentiation/genetics , Extracellular Matrix/metabolism , Signal Transduction/physiologyABSTRACT
Citrate is a critical metabolic substrate and key regulator of energy metabolism in mammalian cells. It has been known for decades that the skeleton contains most (>85%) of the body's citrate, but the question of why and how this metabolite should be partitioned in bone has received singularly little attention. Here, we show that osteoblasts use a specialized metabolic pathway to regulate uptake, endogenous production, and the deposition of citrate into bone. Osteoblasts express high levels of the membranous Na+-dependent citrate transporter solute carrier family 13 member 5 (Slc13a5) gene. Inhibition or genetic disruption of Slc13a5 reduced osteogenic citrate uptake and disrupted mineral nodule formation. Bones from mice lacking Slc13a5 globally, or selectively in osteoblasts, showed equivalent reductions in cortical thickness, with similarly compromised mechanical strength. Surprisingly, citrate content in mineral from Slc13a5-/- osteoblasts was increased fourfold relative to controls, suggesting the engagement of compensatory mechanisms to augment endogenous citrate production. Indeed, through the coordinated functioning of the apical membrane citrate transporter SLC13A5 and a mitochondrial zinc transporter protein (ZIP1; encoded by Slc39a1), a mediator of citrate efflux from the tricarboxylic acid cycle, SLC13A5 mediates citrate entry from blood and its activity exerts homeostatic control of cytoplasmic citrate. Intriguingly, Slc13a5-deficient mice also exhibited defective tooth enamel and dentin formation, a clinical feature, which we show is recapitulated in primary teeth from children with SLC13A5 mutations. Together, our results reveal the components of an osteoblast metabolic pathway, which affects bone strength by regulating citrate deposition into mineral hydroxyapatite.
Subject(s)
Citric Acid , Symporters , Animals , Mice , Citric Acid/metabolism , Symporters/metabolism , Durapatite/metabolism , Citrates , Citric Acid Cycle , Osteoblasts/metabolism , Mammals/metabolism , Dicarboxylic Acid Transporters/metabolismABSTRACT
After injury, a cascade of events repairs the damaged tissue, including expansion and differentiation of the progenitor pool and redeposition of matrix. To guide future wound regeneration strategies, we compared single-cell sequencing of regenerative (third phalangeal element [P3]) and fibrotic (second phalangeal element [P2]) digit tip amputation (DTA) models as well as traumatic heterotopic ossification (HO; aberrant). Analyses point to a common initial response to injury, including expansion of progenitors, redeposition of matrix, and activation of transforming growth factor ß (TGF-ß) and WNT pathways. Surprisingly, fibrotic P2 DTA showed greater transcriptional similarity to HO than to regenerative P3 DTA, suggesting that gene expression more strongly correlates with healing outcome than with injury type or cell origin. Differential analysis and immunostaining revealed altered activation of inflammatory pathways, such as the complement pathway, in the progenitor cells. These data suggests that common pathways are activated in response to damage but are fine tuned within each injury. Modulating these pathways may shift the balance toward regenerative outcomes.
Subject(s)
Bone and Bones , Musculoskeletal System , Ossification, Heterotopic , Regeneration , Amputation, Surgical , Bone and Bones/injuries , Cell Differentiation , Humans , Musculoskeletal System/injuries , Transforming Growth Factor betaABSTRACT
Sarcomas produce an abnormal extracellular matrix (ECM), which in turn provides instructive cues for cell growth and invasion. Neural EGF like-like molecule 1 (NELL1) is a secreted glycoprotein characterized by its nonneoplastic osteoinductive effects, yet it is highly expressed in skeletal sarcomas. Here, we show that genetic deletion of NELL1 markedly reduces invasive behavior across human osteosarcoma (OS) cell lines. NELL1 deletion resulted in reduced OS disease progression, inhibiting metastasis and improving survival in a xenograft mouse model. These observations were recapitulated with Nell1 conditional knockout in mouse models of p53/Rb-driven sarcomagenesis, which reduced tumor frequency and extended tumor-free survival. Transcriptomic and phosphoproteomic analyses demonstrated that NELL1 loss skews the expression of matricellular proteins associated with reduced FAK signaling. Culturing NELL1 knockout sarcoma cells on wild-type OS-enriched matricellular proteins reversed the phenotypic and signaling changes induced by NELL1 deficiency. In sarcoma patients, high expression of NELL1 correlated with decreased overall survival. These findings in mouse and human models suggest that NELL1 expression alters the sarcoma ECM, thereby modulating cellular invasive potential and prognosis. Disruption of NELL1 signaling may represent a novel therapeutic approach to short-circuit sarcoma disease progression. SIGNIFICANCE: NELL1 modulates the sarcoma matrisome to promote tumor growth, invasion, and metastasis, identifying the matrix-associated protein as an orchestrator of cell-ECM interactions in sarcomagenesis and disease progression.
Subject(s)
Calcium-Binding Proteins , Osteosarcoma , Sarcoma , Animals , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Disease Progression , Extracellular Matrix/metabolism , Humans , Mice , Osteosarcoma/genetics , Osteosarcoma/metabolism , Sarcoma/metabolismABSTRACT
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.
ABSTRACT
De novo limb regeneration after amputation is restricted in mammals to the distal digit tip. Central to this regenerative process is the blastema, a heterogeneous population of lineage-restricted, dedifferentiated cells that ultimately orchestrates regeneration of the amputated bone and surrounding soft tissue. To investigate skeletal regeneration, we made use of spatial transcriptomics to characterize the transcriptional profile specifically within the blastema. Using this technique, we generated a gene signature with high specificity for the blastema in both our spatial data, as well as other previously published single-cell RNA-sequencing transcriptomic studies. To elucidate potential mechanisms distinguishing regenerative from non-regenerative healing, we applied spatial transcriptomics to an aging model. Consistent with other forms of repair, our digit amputation mouse model showed a significant impairment in regeneration in aged mice. Contrasting young and aged mice, spatial analysis revealed a metabolic shift in aged blastema associated with an increased bioenergetic requirement. This enhanced metabolic turnover was associated with increased hypoxia and angiogenic signaling, leading to excessive vascularization and altered regenerated bone architecture in aged mice. Administration of the metabolite oxaloacetate decreased the oxygen consumption rate of the aged blastema and increased WNT signaling, leading to enhanced in vivo bone regeneration. Thus, targeting cell metabolism may be a promising strategy to mitigate aging-induced declines in tissue regeneration.
Subject(s)
Extremities , Transcriptome , Amputation, Surgical , Animals , Bone Regeneration/genetics , Bone and Bones , Mammals , Mice , Wound HealingABSTRACT
Bone regeneration following injury is initiated by inflammatory signals and occurs in association with infiltration by sensory nerve fibers. Together, these events are believed to coordinate angiogenesis and tissue reprogramming, but the mechanism of coupling immune signals to reinnervation and osteogenesis is unknown. Here, we found that nerve growth factor (NGF) is expressed following cranial bone injury and signals via p75 in resident mesenchymal osteogenic precursors to affect their migration into the damaged tissue. Mice lacking Ngf in myeloid cells demonstrated reduced migration of osteogenic precursors to the injury site with consequently delayed bone healing. These features were phenocopied by mice lacking p75 in Pdgfra+ osteoblast precursors. Single-cell transcriptomics identified mesenchymal subpopulations with potential roles in cell migration and immune response, altered in the context of p75 deletion. Together, these results identify the role of p75 signaling pathway in coordinating skeletal cell migration during early bone repair.
Subject(s)
Nerve Growth Factor , Receptors, Nerve Growth Factor , Signal Transduction , Animals , Cell Movement , Mice , Nerve Growth Factor/metabolism , Osteoblasts/metabolism , Osteogenesis/genetics , Receptors, Nerve Growth Factor/metabolismABSTRACT
The outer coverings of the skeleton, which is also known as the periosteum, are arranged in concentric layers and act as a reservoir for tissue-specific bone progenitors. The cellular heterogeneity within this tissue depot is being increasingly recognized. Here, inducible PDGFRα reporter animals were found to mark a population of cells within the periosteum that act as a stem cell reservoir for periosteal appositional bone formation and fracture repair. During these processes, PDGFRα reporter+ progenitors give rise to Nestin+ periosteal cells before becoming osteoblasts and osteocytes. The diphtheria toxin-mediated ablation of PDGFRα reporter+ cells led to deficits in cortical bone formation during homeostasis and a diminutive hard callus during fracture repair. After ossicle transplantation, both mouse PDGFRα reporter+ periosteal cells and human Pdgfrα+ periosteal progenitors expand, ossify, and recruit marrow to a greater extent than their counterpart periosteal cells, whereas PDGFRα reporter- periosteal cells exhibit a predisposition to chondrogenesis in vitro. Total RNA sequencing identified enrichment of the secreted factors Fermt3 and Ptpn6 within PDGFRα reporter+ periosteal cells, which partly underlie the osteoblastogenic features of this cell population.